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Targeting mTOR for cancer therapy

Targeting mTOR for cancer therapy Mechanistic target of rapamycin (mTOR) is a protein kinase regulating cell growth, survival, metabolism, and immunity. mTOR is usually assembled into several complexes such as mTOR complex 1/2 (mTORC1/2). In cooperation with raptor, rictor, LST8, and mSin1, key components in mTORC1 or mTORC2, mTOR catalyzes the phosphorylation of multiple targetssuchasribosomal proteinS6kinase β-1 (S6K1), eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), Akt, protein kinase C (PKC), and type-I insulin-like growth factor receptor (IGF-IR), thereby regulating protein synthesis, nutrients metabolism, growth factor signaling, cell growth, and migration. Activation of mTOR promotes tumor growth and metastasis. Many mTOR inhibitors have been developed to treat cancer. While some of the mTOR inhibitors have been approved to treat human cancer, more mTOR inhibitors are being evaluated in clinical trials. Here, we update recent advances in exploring mTOR signaling and the development of mTOR inhibitors for cancer therapy. In addition, we discuss the mechanisms underlying the resistance to mTOR inhibitors in cancer cells. Keywords: Cancer, Drug resistance, mTOR, Oncogene, Targeted therapy Introduction mTORC3, consists of ETV7, mTOR, and other undefined The mechanistic target of rapamycin (mTOR) is a dual- components [3]. mTORC1 senses nutrients, growth factors, specificity protein kinase phosphorylating serine/threo- and cellular energy to orchestrate nucleotide, lipid, and nine as well as tyrosine residues [1]. Since the catalytic protein synthesis; inhibit autophagy; and stimulate cell domain of mTOR resembles that of lipid kinases such as growth [2]. mTORC2 is not only regulated by growth phosphoinositide 3-kinase (PI3K), mTOR is considered as factors, but also activates type I insulin-like growth factor an atypical protein kinase belonging to the PI3K-related receptor (IGF-IR) and insulin receptor (InsR) through the kinase family [2]. As a core component of several distinct tyrosine kinase activity of mTOR [1]. Besides, mTORC2 complexes including mTOR complex 1 (mTORC1), regulates the actin polarization and endocytosis [4, 5]. mTOR complex 2 (mTORC2), and a putative mTOR The mTOR signaling pathway has critical roles in complex 3 (mTORC3), mTOR has critical roles in diverse mammalian metabolism and physiology. The de-regulated biological processes, such as cell proliferation, survival, activity of mTOR is involved in many pathophysiological autophagy, metabolism, and immunity [2, 3]. While conditions, such as aging, Alzheimer’s disease, diabetes, mTOR and mammalian lethal with SEC13 protein 8 obesity, and cancer [2]. As a natural inhibitor of (mLST8) are common members of both mTORC1 and mTORC1, rapamycin is able to increase lifespan in mice mTORC2, regulatory-associated protein of mTOR (raptor), [6, 7]. mTOR activity is frequently de-regulated in a the 40 kDa proline-rich Akt substrate (PRAS40), and DEP variety of human cancers, such as breast, prostate, lung, domain-containing protein 6 (DEPTOR) are specific liver, and renal carcinomas. Upregulation of mTOR signa- members of mTORC1 [1, 2]. Instead, rapamycin-insensitive ling can promote tumor growth and progression through companion of mTOR (rictor) and mammalian stress- diverse mechanisms including the promotion of growth activated protein kinase-interactingprotein1(mSIN1or factor receptor signaling, angiogenesis, glyolytic meta- MAPKAP1) are unique components in mTORC2 but not bolism, lipid metabolism, cancer cell migration, and mTORC1 [1]. Another rapamycin-insensitive complex, suppression of autophagy [1, 2]. Hence, mTOR is a promising target for cancer therapy. In this review, we discuss the roles of mTOR in human cancer and * Correspondence: jyangfu@scu.edu.cn the rationales and challenges for developing mTOR Laboratory of Oncogene, Cancer Center, West China Hospital, Sichuan inhibitors to treat cancer. University, Chengdu, China Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 2 of 19 The assembly of mTOR complexes may stabilize the dimer by binding the HEAT repeats 11– The studies of mTORC1 structure demonstrate that 13 in one mTOR and repeats 20–22 in another mTOR [8, mTORC1 adopts a dimeric architecture with an overall 11]. In addition, raptor is required for recruiting substrates size of (280~300) × (200~210) × (100~130) Å [8, 9]. to mTORC1 [12, 13]. Both mTOR and raptor are sub- mTOR and LST8 form the core of mTOR complex that jected to phosphorylation at multiple residues (Fig. 1a), contains raptor and other regulatory proteins [8]. The which positively or negatively regulates mTORC1 activity. human mTOR contains 2549 amino acids that form TheassemblyofmTORC2and Saccharomyces cerevisiae several domains including the NH -terminal HEAT TORC2 follows a similar principle to mTORC1. The (N-HEAT), middle HEAT (M-HEAT), FAT, and kinase human mTORC2 structure reveals a hollow rhombohedral domain with a FRB insertion (Fig. 1). Raptor also fold with overall dimensions of ~ 220 × 200 × 130 (Å ) contains a HEAT domain, as well as WD40 and cas- [14]. A dimer of mTOR is located in the core of this pase-like domain [8, 9]. Besides, LST8 has WD40 domain. complex, while each mTOR or TOR heterodimerizes The HEAT motifs have conserved Asp and Arg residues with rictor and mSIN1 [14, 15]. Rictor has an NH -ter- at positions 19 and 25, respectively. A signature motif of minal armadillo (ARM) repeat cluster (~ 900 residues), WD40 repeats is ~ 40 amino acids often ending with a and the rest of the rictor is largely unstructured (Fig. 1b) tryptophan-aspartic acid (W-D) dipeptide [10]. The [16]. Interestingly, ARM and HEAT domains have similar HEAT repeats 12–13 in one mTOR interact with the conserved residues that form the hydrophobic domain HEAT repeats 20–23 in the M-HEAT domain of an- core and may have a common phylogenetic origin [17]. In other mTOR, thereby forming a dimer [8]. Raptor addition, mSin1 has a CRIM, a Ras-binding domain Fig. 1 The domains in key components of mTORC1 and mTORC2. a The molecular weight, domains, and phosphorylation sites in key components of mTORC1, including mTOR, LST8, and raptor. b The molecular weight, domains, and phosphorylation sites in key components of mTORC2, including mTOR, mSin1, and rictor Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 3 of 19 (RBD), and a pleckstrin homology (PH) domain [18]. an increase in AMP/ATP ratio, which activates the energy During the assembly of mTORC2, the FRB domain of sensor AMP-dependent kinase (AMPK). AMPK stimu- mTOR binds to mSin1 and the carboxy terminal region of lates the GAP activity of TSC and then promotes the rictor, while the NH -terminal portion (residues 506–516) inhibition of RHEB by TSC, leading to the downregulation of rictor interacts with the COOH-terminal region (resi- of mTORC1 [28]. In addition, the TCA cycle metabolite dues 1186-1218) of M-HEAT of mTOR [14]. In addition, ketoglutarate inhibits mTORC1 through repressing ATP mSin1 directly binds to rictor. Both rictor and mSin1 are synthase, increasing AMP/ATP ratio and activating responsible for recruiting substrates to mTORC2. Of note, AMPK [29]. Cellular energy deficiency usually leads to both rictor and mSin1 have mTOR-independent partners. endoplasmic reticulum stress, which in turn induces the For example, rictor interacts with integrin-linked kinase unfolded protein response (UPR). Ire1, ATF6, and PERK and promotes its phosphorylation of Akt [19], while are three major mediators of the UPR. Upon ER stress, mSin1 interacts with Ras and inhibits ERK1/2 phosphory- ATF6 can induce RHEB expression, which in turn lation [20]. Thus, the outcome from the manipulation of promotes mTORC1 activation and cell survival [30]. rictor or mSin1 alone may not exactly reflect the function However, overactivated mTORC1 is also harmful to of mTORC2. cell survival under ER stress. Mutations in TSC1/2 or activation of RHEB renders cells hypersensitive to ER Regulation of mTORC1 activity stress-induced apoptosis, which may be due to the The activity of mTORC1 is regulated by growth factors, downregulation of ATF4/6 by mTOR [31]. Therefore, cellular energy, stresses and nucleotides, etc. The lyso- mTORC1 may have versatile effects on cell survival somes are primary sites for mTORC1 activation. The under ER stress. activation of mTORC1 by growth factors is dependent on While the regulation of mTORC1 by growth factors is Ras homolog enriched in the brain (RHEB), a lysosomal dependent on RHEB and the TSC complex, amino acids GTPase that directly interacts with mTOR and activates it can stimulate mTORC1 independent of TSC. The regula- [21]. Upon binding to growth factors such as epidermal tion of mTORC1 by amino acids is very complicated, growth factor (EGF) and insulin-like growth factor (IGF), involving multiple amino acid sensors and protein the growth factor receptors (EGFR, IGFR, etc.) are machinery [32]. The lysosomal Ragulator (RAG) guano- activated, which in turn activate PI3K-PDK1-Akt signaling sine triphosphatases (GTPases) play key roles in the pathway. Active Akt phosphorylates tuberous sclerosis activation of mTORC1 by amino acids. RAGA or RAGB complex 2 (TSC2) and inhibits the TSC complex, a heterodimerizes with RAGC or RAGD [33]. Further, RAG GTPase-activating protein (GAP) complex consisting of proteins form a large complex with LAMTOR1/2/3/4/5, TSC1/2 and TRE2-BUB2-CDC16 domain family member which recruit RAG and mTORC1 to the lysosomal surface 7 (TBC1D7) [22, 23]. The TSC complex can inactivate [34]. The activity of RAG is regulated by two complexes, RHEB thereby inhibiting mTOR [24]. Therefore, the GATOR1 and GATOR2. GATOR1, which is composed of activation of Akt leads to the depression of RHEB and DEPDC5, NPRL2, and NPRL3, inhibits the GTPase- then activates mTORC1. Moreover, the ubiquitination of activated protein (GAP) activity of RAGA/B thereby RHEB regulates its ability to activate mTORC1 [21]. The repressing the activation of mTORC1 by amino acids [35]. E3 ubiquitin ligase RNF152 catalyzes RHEB ubiquitina- Instead, GATOR2, a protein complex consisting of tion, leading to an increase in the interaction between MIOS, WDR24, WDR59 SEH1L, and SECB, negatively RHEB and TSC [21]. In contrast, Akt can phosphorylate regulates GATOR1 by inducing DEPDC5 degradation the deubiquitinase USP4 that promotes RHEB deubiquiti- [35]. Furthermore, KICSTOR, a large complex consisting of nation thereby releasing RHEB from TSC [21]. KPTN, ITFG2, C12ORF66, and seizure threshold 2 (SZT2), Downstream of the growth factor receptors, the recruits GATOR1 to the lysosomal surface and mediates mitogen-activated protein kinase (MAPK) also up- the interaction between GATOR1 and RAG [36, 37]. regulates mTORC1 activity. Mechanistically, MEK1/2 Sestrin (SESN) is another category of negative inhibitors promotes raptor phosphorylation through ERK1/2 and of amino acid-induced mTORC1 activation. Mechanis- p90 ribosomal S6 kinase (RSK1/2). ERK1/2 directly tically, SESNs interact with GATOR2, leading to the phosphorylates raptor at S8, S696, and S863, while release of GATOR1 from GATOR2. The released RSK1/2 phosphorylates raptor at S719/722 [25, 26]. GATOR1 in turn inhibits RAG and mTORC1 [38–40]. Of Meanwhile, the intestinal cell kinase (ICK), a MAPK- note, SESN2 is known as a leucine sensor in mTORC1 related kinase, phosphorylates raptor at T908 [27]. signaling. Leucine directly binds to SESN2, leading to Phosphorylation of raptor by ERK/RSK/ICK promotes the dissociation of SESN2 from GATOR2. The re- the activation of mTORC1. leased GATOR2 binds to GATOR1 and then prevents the mTORC1 not only senses growth factors, but also inhibition of RAG by GATOR1. These sequential pro- responds to cellular energy. Low cellular energy results in cesses result in RAG-mediated mTORC1 activation [41]. Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 4 of 19 To prevent the overactivation of mTORC1 by amino How does mTORC2 respond to cellular energy and nu- acids, there are negative feedback pathways to RAG- trients? The energy sensor AMPK inhibits mTORC1 and mediated mTORC1 activation. Two E3 ubiquitin then releases the suppression of mTORC2 by mTORC1, ligases, RNF152 and SKP2, reportedly induce RAGA leading to the activation of mTORC2 [56]. Thus, upregu- ubiquitination and potentiate the binding of RAGA to lation of mTORC2 may help cells adapt to low levels of GATOR1 [42, 43]. While leucine sufficiency is sensed cellular energy. Moreover, mTORC2 is activated by by SESN2, the stimulation of mTORC1 by arginine is glutamine starvation. Activated mTORC2 upregulates the mediated by SLC38A9 [44]. Moreover, the ubiquitin expression and phosphorylation of glutamine:fructose-6- ligase TRAF6 can catalyze K63 ubiquitination of both phosphate amidotransferase 1 (GFAT1), the rate-limiting Akt and mTOR thereby promoting the activation of enzyme of the hexosamine biosynthesis pathway (HBP) Akt and mTORC1 by amino acids [45, 46]. [57, 58]. A study of budding yeast demonstrates that the In addition, mTOR may be activated by lipid and LKB1-ELM1-GIN4/HSL1 axis is required for coordinating cholesterol. Fatty acid metabolism leads to the de novo TORC2 signaling to the changes in carbon source [59]. It synthesis of phosphatidic acid (PA), which stabilizes both remains to know if similar pathway works in human mTORC1 and mTORC2 [47]. Moreover, cholesterol can cancer cells. stimulate mTORC1 activation and growth signaling. Similar to mTORC1, mTORC2 is also stabilized by Mechanistically, SLC38A9 acts as a lysosomal choles- phosphatidic acid (PA), a central metabolite in the synthe- terol sensor to stimulate the activation of mTORC1 by sis of membrane phospholipids [60]. The generation of PA RAG complex [48]. Recently, it was reported that is catalyzed by the phospholipase D, diacylglycerol kinases, mTORC1 is also responsive to the levels of purine and lysophosphatidic acid acyltransferases. Moreover, the nucleotides [49]. While adenylate stimulates mTORC1 activity of mTORC1 and mTORC2 is regulated by mLST8 by inhibiting TSC, guanylate downregulates RHEB and ubiquitination. It has been reported that the E3 ubiquitin then inhibits mTORC1 [49]. The mechanisms under- ligase TRAF2 positively regulates K63-linked polyubiquiti- lying the regulation of TSC and RHEB by adenylate and nation of mLST8, which impairs its interaction with guanylate remain to be known. mSin1 and compromises the mTORC2 integrity, but enhances the assembly of mTORC1 [61]. On the contrary, Regulation of mTORC2 activity the deubiquitinase OTUDB7 removes polyubiquitin Although mTORC1 and mTORC2 are distinct com- chains from G L to promote G L interaction with mSin1 β β plexes, there is a crosstalk between these two complexes. and the integrity of mTORC2 [61]. Besides, the exchange On one hand, mTORC2 can activate IGF-IR-Akt axis factor found in platelets, leukemic, and neuronal tis- thereby upregulating mTORC1 [1]. On the other hand, sues (XPLN) interacts with mTORC2 and negatively mTORC1 feeds back to inhibit mTORC2 via S6K1, one regulates mTORC2 activity [62]. Lastly, mTOR is a of the substrates of mTORC1. Once activated by target of proteasomal degradation when it is ubiquiti- mTORC1, S6K1 phosphorylates rictor and mSin1 on nated by FBXW7 [63]. T1135 and T86/398, respectively, leading to the impair- ment of mTORC2 integrity [50–52]. Targets of mTORC1 and mTORC2 While mTORC2 directly activates IGF-IR and InsR, As a protein kinase, mTOR catalyzes the phosphorylation receptor tyrosine kinases such as EGFR, PDGFR, and of its targets and regulates their activity. mTORC1 and IGF-IR can activate mTORC2 via PI3K. Mechanistically, mTORC2 have different substrates. While the repertoire PI3K-induced PtdIns (3,4,5) P3 (PIP3) binds to the PH of mTOR substrates keeps increasing, there are more domain of mSin1 and then disables the inhibition of targets remaining to be identified. S6K1 and 4E-BP1 are mTOR kinase domain by mSin1, thereby activating two well-known mTORC1 targets. mTORC1 phosphory- mTORC2 [18]. In addition, PI3K promotes the asso- lates S6K1 at T389 and 4E-BP1 at multiple residues [64]. ciation of mTORC2 with ribosome, where mTORC2 is Phosphorylation of S6K1 by mTORC1 leads to increased activated [53]. Therefore, mTORC2 also responds to protein and nucleotide synthesis. While 4E-BP1 is a nega- growth factors. Notably, another study suggests that tive regulator of 5′cap-dependent mRNA translation, mTORC2 activity is localized in the plasma membrane, phosphorylation of 4E-BP1 by mTORC1 induces its mitochondria, and endosomal vesicles, and the activity dissociation from eIF4E, thereby relieving its inhibition of of mTORC2 via the mSin1-PH domain at the plasma protein synthesis [65]. To cope with increased protein membrane is PI3K- and growth factor-independent [54]. synthesis, mTORC1 also promote ribosome biogenesis by In addition, IKKα interacts with mTORC2 and enhances inducing ribosomal RNA transcription. Mechanistically, its kinase activity towards Akt [55]. These data suggest mTORC1 may translocate to the nucleus, where it binds that the activation of mTORC2 involves multiple to ribosomal DNA promoter [66–68]. Nuclear mTOR also location and different mechanisms. phosphorylates TFIIIC and Maf1, thereby promoting Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 5 of 19 tRNA gene transcription [69]. In fact, nuclear mTOR reg- various kinds of cancer, such as breast cancer, gastric can- ulates RNA polymerase 1/2/3-driven transcription. In cer, and liver cancer [78, 79]. Moreover, rictor is overex- addition, mTORC1 phosphorylates the E3 ubiquitin ligase pressed in human cancers of the brain, breast, lung, gastric, SKP2 at S64 and then inhibits SKP2 ubiquitination and colon, liver, and tongue [80, 81]. degradation [70]. Given that SKP2 promotes the de- Given that mTOR has critical roles in tumor progres- gradation of many proteins, mTORC1 may regulate the sion, mTOR inhibitors hold promise in cancer therapy. turnover of SKP2 substrates indirectly. Thus, mTORC1 Indeed, rapamycin analogs (rapalog) have been approved not only promotes protein synthesis, but also regulates for treating cancer in the clinic. In addition, many protein degradation. mTOR inhibitors with different mechanisms of action Following the identification of mTORC2, it was found have been developed, some of which are undergoing that protein kinase C (PKC) α/β were the substrates of clinical trials in variety types of human cancer. mTORC2 that regulates the actin cytoskeleton [4, 71]. Moreover, mTORC2 phosphorylates and activates other Rapalog AGC kinases, such as serum and glucocorticoid-induced Rapamycin was originally identified as an antifungal, kinase (SGK) and Akt. mTORC2 phosphorylates Akt at immunosuppressive, and antiproliferative agent. Later S473, leading to allosteric activation of Akt in cooperation studies revealed that rapamycin binds to the 12 kDa with the catalytic activation by PDK1, which phosphory- FK506-binding protein (FKBP12) and then inhibits lates Akt at T308 [72]. During the synthesis of nascent mTORC1 [82]. Since rapamycin has poor solubility proteins, mTORC2 can co-translationally phosphorylate and pharmacokinetics, it is not suitable for treating some polypeptides while they are attached to the ribo- human cancer. So far, several water-soluble rapamycin some. IGF2 mRNA-binding protein (IMP) is responsible analogs have been developed. For example, temsiroli- for the splicing and translation of IGF2 mRNA. mTORC2 mus and everolimus exhibit tumor-suppressive effects co-translationally phosphorylates IMP1 at S181 and then in vivo. Both temsirolimus and everolimus have been promotes IMP1 binding to the untranslated region of used to treat advanced renal cell carcinoma (RCC) in IGF2 mRNA and enables translational initiation by the clinic. Moreover, everolimus is prescribed for internal ribosomal entry [73]. mTORC2 not only enhances treating pancreatic neuroendocrine tumors and advanced the production of IGF2 protein, but also phosphorylates breast cancer [83]. Besides, there are many clinical trials and activates IGF-IR and insulin receptor [1]. In contrast to evaluate the efficacy of rapalogs in treating other to mTORC1’s activity as a ser/thr kinase, mTORC2 has types of human cancer, such as advanced gastric tyrosine kinase activity towards IGF-IR/InsR [1]. cancer, hepatocellular carcinoma, non-small cell lung cancer, endometrial cancer, and mantle cell lymphoma mTOR inhibitors for cancer therapy (clinicaltrials.gov). The activity of mTOR is frequently upregulated in Of particular note, the effect of rapalog monotherapy human cancer. The aberrant activation of mTOR in human on solid tumors is modest in the clinic. The incomplete cancer may be attributed to mTOR pathway-activating inhibition of mTOR by rapalogs may result in limited mutations, amplification, or overexpression of the com- clinical success. On the other hand, inhibition of ponents of mTOR complexes and mutations or loss of mTORC1 may lead to feedback activation of IGF-IR and negative regulators of mTOR. PIK3CA mutations are Akt, which compromises the anti-cancer effect of frequently detected in human cancer. Activation of PI3K rapalogs [1]. Taking into account the complexity of promotes both mTORC1 and mTORC2 activation. In mTOR signaling networks, it is not hard to understand addition, mutations in KRAS and BRAF may lead to that the response to rapalogs varies in patients with mTORC1 activation. Especially, KRAS can directly bind to cancer, such as metastatic RCC. It is desirable that there PIK3CA (p110α) and activates PI3K pathway, leading to are biomarkers to predict the responses to mTOR in- mTOR activation [74]. mTOR-activating mutations are ob- hibition. KRAS, BRAF, and TSC mutations are known as served in kidney cancer. While mTOR activity is usually resistant markers for mTOR inhibitors, whereas PIK3CA upregulated by growth factors and amino acids, activating mutations are sensitive marker [84, 85]. However, the mutations in mTOR may result in RAG- and RHEB- roles of TSC1/2 and mTOR mutations in responding to independent mTOR hyperactivation, thus loss of the de- rapalogs remain controversial. Although it has been pendency on growth factors and amino acids [75]. Point reported that mutations in TSC1/2 and mTOR are more mutations in RHEB and GATOR1 were also detected in frequent in RCC patients who respond well to rapalogs, renal cancer and endometrial cancer [76]. RHEB1 is over- the majority of rapalog responders have no mutations in expressed in acute myeloid leukemia (AML) and promotes mTOR pathway, suggesting that other factors are also AML progression [77]. Whereas mTOR amplification is involved in rapalog sensitivity [86]. Notably, rapalogs rare in human cancer, rictor amplification is detected in usually arrest cell proliferation but does not induce Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 6 of 19 apoptosis. Despite the initial response, tumors frequently volume in cisplatin-resistant ovarian cancer model [104]. develop resistance to these agents. Similar to PP242, AZD2014 enhances the radiosensitivity of glioblastoma stem-like cells [105]. Based on the above- ATP-competitive mTOR inhibitors described studies, it appears that the pan-mTORC1/2 in- To more completely inhibit mTOR, a number of ATP- hibitors generally reverse rapalog resistance, endocrine re- competitive mTOR inhibitors have been developed to sistance, chemoresistance, and radioresistance. target both mTORC1 and mTORC2. Tumors that are addicted to the mTOR signaling pathway may be Dual PI3K/mTOR inhibitors sensitive to this kind of inhibitors. Unlike rapalogs, ATP- Although inhibition of mTORC1 and mTORC2 can competitive mTOR inhibitors can not only arrest cell downregulate Akt S473 phosphorylation, mTOR inhibi- growth, but also induce apoptosis. MLN0128 (also called tors may paradoxically enhance the PI3K/PDK1 axis. INK128, sapanisertib, TAK-228) is a pan-mTOR in- Thus, an inhibitor targeting both PI3K and mTOR may hibitor that has potent in vitro and in vivo anti-tumor have better anti-cancer activity compared to targeting effects, and has underwent clinical trials for solid tumors mTOR alone [106, 107]. Due to the similarity between such as bone and soft tissue sarcoma, breast cancer, and PI3K and mTOR, some chemicals can inhibit both PI3K primary effusion lymphoma, a non-Hodgkin B cell and mTOR. NVP-BEZ235 (dactolisib) inhibits the activity lymphoma that usually results from infection of Kaposi of multiple class I PI3K isoforms, mTOR and ataxia sarcoma-associated herpesvirus [87–90]. MLN0128 also telangiectasia, and Rad3-related protein (ATR) and has reduces tumor growth in CD44-high HCC xenografts potent anti-cancer activity [108]. Notably, NVP-BEZ235 and resensitizes HCC to sorafenib [91]. Of note, can penetrate the blood-brain barrier after systemic MLN0128 is an effective agent even in tumors that are administration [109]. Therefore, it can be used to treat resistant to rapamycin or chemotherapy. A recent study glioma and reverse temozolomide resistance [110]. In demonstrates that MLN0128 can overcome resistance to addition, NVP-BEZ235 can suppress paclitaxel-resistant everolimus and reduce tumor size by 20% in PIK3CA- gastric cancer, which exhibits increased PI3K/mTOR mutant colorectal cancers [92]. In addition, MLN0128 activity [111]. can induce tumor shrinkage in patient-derived xenograft LY3023414, a complex fused imidazoquinolinone, is model of pancreatic neuroendocrine tumors, even in an oral PI3K/mTOR and DNA-PK inhibitor that has everolimus-resistant tumors [93]. anti-tumor effects in animal models. Combination of PP242 (Tokinib) is another selective ATP-competitive LY3023414 with standard chemotherapeutic drugs has inhibitor of mTOR that has a promising anti-cancer additive anti-tumor activity [112, 113]. Another dual activity over several cancer types, such as leukemia, PI3K/mTOR inhibitor voxtalisib (SAR245409, XL765), a gastric cancer, and colon cancer [94, 95]. Given that pyridopyrimidinone derivative, significantly inhibits tumor the Akt-mTOR signaling pathway is upregulated in growth in multiple human xenograft models [114]. Com- platinum-resistant cancer cells, studies demonstrate bination of voxtalisib and the MEK inhibitor pimasertib that mTORC1/2 inhibitor, such as PP242 and MLN0128, synergistically inhibits certain endometrial cancer cells can re-sensitize platinum-resistant ovarian cancer cells to growth [115]. Other dual PI3K/mTOR inhibitors include carboplatin in vitro and in vivo [96, 97]. Mechanistically, PQR309, XH00230381967, SN20229799306, GSK2126458 mTOR inhibition leads to a sharp decrease in the transla- (omipalisib), and PKI-587. tion of DNA damage and repair response and pro-survival Of note, PQR309 is a 4,6-dimorpholino-1,3,5-triazine- mRNAs, including CHK1 [98]. Consistent with the in- based, brain-penetrant, and orally bioavailable PI3K/ hibition of DNA repair, mTOR inhibitors are also effective mTOR inhibitor [116]. PQR309 effectively inhibits lym- in enhancing radiosensitivity or restoring radiosensitivity phoma in monotherapy and in combination therapy in radioresistant tumors [99, 100]. Moreover, inhibition of with other drugs, such as the BCL2 inhibitor veneto- mTORC1/C2 signaling improves anti-leukemia efficacy of clax, the HDAC inhibitor panobinostat, the Bruton’s JAK/STAT blockade in CRLF2-rearranged and/or JAK- tyrosine kinase inhibitor ibrutinib, lenalidomide, the driven Philadelphia chromosome-like acute B cell lympho- BET proteolysis-targeting chimera ARV-825, the prote- blastic leukemia [101]. asome inhibitor marizomib, and the anti-CD20 mono- Both AZD2014 (vistusertib) and its analog AZD8055, clonal antibody rituximab [117]. Moreover, PQR309 can two ATP-competitive mTORC1/2 inhibitors, are highly suppress cancer cells with primary or secondary resistance effective in treating estrogen receptor (ER)-positive breast to the PI3Kδ. PQR620 and the PI3K/mTORC1/2 inhibitor cancer. Moreover, AZD2014 and AZD8055 can suppress PQR530 effectively cross the blood-brain barrier [118]. breast cancer with acquired resistance to endocrine The dual specificity PI3K/mTOR inhibitor gedatolisib therapy, rapalogs, and paclitaxel [102, 103]. In addition, a (PKI-587, PF05212384) is a bis(morpholino-1,3,5-tria- combination of AZD2014 with paclitaxel reduces tumor zine) derivative [119]. Gedatolisib inhibits tumor growth Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 7 of 19 in breast, colon, lung, and glioma xenograft models and Principle mechanisms of mTOR inhibitor resistance displays efficacy against T cell acute lymphoblastic in cancer leukemia (T-ALL) and Philadelphia chromosome (Ph)- Drug resistance is a serious problem in treating cancer. like B cell acute lymphoblastic leukemia (Ph-like ALL) Although there may be an initial response, long-lasting [107, 120]. Combination of gedatolisib with ruxolitinib treatment with chemotherapeutic or molecular-targeted or dasatinib has superior efficacy than a single agent in drugs often faces the challenge of drug resistance. Due to CRLF2/JAK-mutant models and ABL/PDGFR-mutant the tumor heterogeneity, some tumors do not respond to models, respectively [120]. In addition, gedatolisib a given drug at all. Clonal selection, adaptive evolution, sensitizes head, neck, and nasophageal carcinoma to ra- and resistance to cell death are general mechanisms for diation therapy [121, 122] and sensitizes EGFR-resistant drug resistance. Due to the complexity and crosstalk in head and neck carcinoma to cetuximab [123]. Thus, signaling networks, cancer cells may adapt to an inhibitor gedatolisib may be a candidate sensitizer to radiotherapy that targets a given signaling pathway via the com- and targeted therapy. pensatory activation of other pathways. Although mTOR GSK2126458 (omipalisib) is an orally bioavailable inhibitors exhibit potent anti-cancer effects in many inhibitor of PI3Kα and mTOR [124]. Omipalisib potently preclinical models, resistance does occur. As described inhibits FGFR4-V550E tumor-derived cell and human below, there are multiple mechanisms underlying the rhabdomyosarcoma cell viability and reduces the growth resistance to mTOR inhibitors (Fig. 2). of rhabdomyosarcoma in vivo [125]. In addition, a combination of the PI3K/mTOR inhibitor VS-5584 and Drug efflux by ATP binding cassette transporters the Wnt inhibitor ICG-001 synergistically inhibits AML ATP-binding cassette (ABC) transporters constitute drug with high PRL-3 expression [126]. Finally, the efficacy efflux pumps that decrease the intracellular levels of of mTOR inhibitor may be enhanced by linking the drugs, leading to poor treatment outcome. Overexpres- kinase inhibitor to rapamycin (RapaLink) [127]. EZH2 sion of ABC transporters is a general mechanism for (Y641X)-mutant lymphomas show increased sensiti- multi-drug resistance in cancer. The same may be true vity to RapaLink-1 [128]. Given that RapaLink in- for mTOR inhibitor resistance. In fact, the mTOR in- tegrates the activity of both rapamycin and mTOR hibitors rapamycin and NVP-BEZ235 are substrates of kinase inhibitor, it is worthwhile looking forward to ABCB1 (P-glycoprotein) and ABCG2 (also called breast the efficacy in clinical trials. Lastly, there are many cancer resistance protein, BCRP), respectively [132]. In drugs that may indirectly inhibit mTOR, such as addition, AZD8055 is transported by both ABCB1 and aspirinand metformin[129–131]. ABCG2 [132]. Fig. 2 The mechanisms for resistance to mTOR inhibitors in cancer cells. ABC transporters, ATP binding cassette transporters; EMT, epithelial-mesenchymal transition Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 8 of 19 Studies show that ABCB1 is overexpressed in luminal FGF1-FGFR-Notch1 axis [147]. Blocking FGFR or Notch1 breast cancer cell lines that are resistant to everolimus may prevent resistance to TORC1/2 inhibitors by abro- [133]. Also, ABCB1 inhibits brain accumulation of gating the expansion of drug-resistant CSCs in TNBC everolimus [134]. Overexpression of ABCG2 in cancer [49]. Moreover, another dual PI3K/mTOR inhibitor PF- cells confers significant resistance to PF-4989216, which 04691502 can induce a stem cell-like gene expression can be reversed by an inhibitor or competitive substrate signature in KRAS-mutant colorectal cancer models of ABCG2 [135]. Moreover, GDC-0980 is subject to [148]. Together, these data suggest that the effects of active efflux by ABCB1 and BCRP, which limits its mTOR inhibitors on CSC may be dependent on the genetic efficacy [136]. The affinity for ABC transporters may background and rewiring of cancer stemness pathways. vary among different mTOR inhibitors. Lowering the affinity for ABC transporters or inhibiting ABC trans- Assembly of the translation machinery porters may enhance the efficacy of mTOR inhibitors. Eukaryotic protein synthesis is regulated by several me- chanisms including cap-dependent and cap-independent Cancer stem cells translation. The cap-dependent pathway involves many Cancer stem cells (CSCs) are a subpopulation in tumor eukaryotic initiation factors (eIF), such as eIF1, eIF2, eIF3, mass that is extremely resistant to standard cancer therapy. eIF4A, eIF4B, eIF4E, eIF4H, eIF5, and eIF6. The protein Slow-cycling CSC is one of the major obstacles to eradicate synthesis is initiated by the association of the 40S ribo- tumor [137]. It is generally thought that the mTOR some subunit with eIF1A and eIF3, followed by binding of pathway is hyperactivated in CSC. Transforming growth the eIF2-GTP-methionine tRNA complex to 40S subunit factor-β (TGF-β) can induce epithelial-mesenchymal tran- and then forming a 43S subunit [149]. The eIF4F complex, sition (EMT), which enhances cancer stem cell generation. which consists of eIF4E, eIF4A, and eIF4G, binds to the mTOR is one of the mediators in TGF-β signaling path- m G cap at the 5′ end of mRNA and then activates ways that enhances cancer stemness and drug resistance mRNA. The activated mRNA is recruited to the 43S com- [138]. The inhibitory effect on CSCs has already been plex and then subjected to ATP-dependent scanning of shown for some mTOR inhibitors [139]. Rapamycin, mRNA to locate the initiating AUG code [150]. Finally, everolimus, and PF-04691502 suppress tamoxifen-induced the 60S ribosome subunit is associated with the 40S sub- activation of breast cancer stem cells [140]. Inhibition of unit to form the 80S initiation complex, possibly assisted mTOR restores tamoxifen resistance in breast cancer cells by eIF5. For the initiation of cap-independent protein [141]. Moreover, the ATP-competitive mTOR inhibitor synthesis, the 40S ribosome subunit binds to an internal Torin1 and PI3K/mTOR inhibitor VS-5584 preferentially region of mRNA, which is referred to as internal ribosome reduce CSC levels in multiple mouse xenograft models of entry sites (IRES), or the untranslated regions of mRNA. human cancer [142, 143]. Given that stimulation of cap-dependent translation is However, the interplay between mTOR inhibitors and one of the major functions of mTORC1, the status of the CSC is complex. Previous studies show that expansion translation machinery and modes of protein translation of CSC promotes the resistance to mTOR inhibitor in may impact on the efficacy of mTOR inhibitors. 4E-BPs leiomyosarcoma [144]. PDK1 signaling toward PLK1- are phosphorylated and inactivated by mTORC1. The MYC activation leads to tumor-initiating cell activation sensitivity to PP242 is correlated with the extent to which and resistance to mTOR inhibition [145]. Inhibition of 4E-BP1 phosphorylation is inhibited by this drug [151]. EZH2, a catalytic component of polycomb repressive Loss of 4E-BPs in tumor cells results in the resistance to complex which plays a critical role in stem cell main- mTOR inhibition. The transcription factor Snail directly tenance, restores sensitivity to PI3K/mTOR pathway represses 4E-BP1 transcription and compromises the anti- inhibition. It appears that the sensitivity to mTOR in- cancer effects of mTOR inhibitors [152]. Of note, Snail is hibitors in CSC may be context- or cell type-dependent. translationally regulated by eIF4E, which is exactly the Of note, one study demonstrates that TP53 mutation target of 4E-BP. Phosphorylation of eIF4E (S209, etc.) and BCL2 phosphorylation affect the sensitivity of promotes Snail synthesis [153]. Therefore, 4E-BP and glioblastoma stem-like cells to mTOR inhibitor [146]. eIF-4E can disable each other. Overexpression of BCL2 (T56/S70) phosphorylation in TP53 wild-type eIF4E or phosphorylation of eIF4E (S209) by MAP glioblastoma stem-like cells is responsible for the lower kinase-interacting kinase 1 (Mnk1/2) leads to a shift from sensitivity to the mTORC1/2 inhibitor AZD8055, as - cap-dependent to cap-independent translation and then compared to TP53-mutated glioblastoma stem-like cells renders cancer cells insensitive to mTOR inhibition [146]. In addition, while mTOR inhibitors reportedly sup- [154, 155]. Thus, inhibition of Mnk1/2 or its up- press CSC, one study demonstrates that treatment of stream kinase ERK1/2 may restore cap-dependent TNBC cell lines with PI3K/mTOR inhibitor or TORC1/2 translation and the sensitivity of mTOR inhibitors inhibitor expands CSC population through upregulating [155]. On the other hand, inhibition of mTORC1 may Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 9 of 19 lead to paradoxical phosphorylation of eIF4E in PI3K- PI3K/Akt/mTOR pathways are tightly involved in tumori- and Mnk-dependent manner and promote cap-independent genesis. While tumors with PIK3CA/PTEN mutations or translation [156]. Hence, a combination of mTOR Akt hyperactivation usually are sensitive to mTOR inhi- and Mnk inhibitors is an effective therapeutic strategy bitors, KRAS/BRAF mutations are predictive biomarkers for cancer [157]. of mTOR inhibitor resistance [148, 166–169]. In addition, Notably, 4E-BP1 is not only phosphorylated by mTORC1, mTOR inhibition may lead to the activation of the but also phosphorylated and inactivated by other kinases MEK-Erk pathway. Combination of RAF/MEK inhibi- such as CDK1, CDK12, and GSK3 . CDK1 can substitute tors and mTOR inhibitors may be a strategy to treat mTORC1 to phosphorylate 4E-BP1 and activate cap- KRAS-mutated cancer [170, 171]. Besides, the activation dependent translation, which is resistant to mTOR of Erk in response to mTOR inhibition can be abrogated inhibition [158]. In addition, CDK12 cooperates with by the CDK4/6 inhibitor palbociclib [172]. Combination mTORC1 to phosphorylate 4E-BP1 and releases it from of CDK4/6 and mTOR inhibitors synergistically inhibits mTORC1 target mRNAs thereby promoting their tumor growth [172, 173]. Alternatively, combined in- translation [159]. Therefore, combinatorial inhibition of hibition of wee1, a protein kinase that regulates the G2 mTOR and CDK1/12 may be synthetically lethal to cancer checkpoint in the cell cycle, with mTOR inhibition may cells. Furthermore, GSK3β can directly phosphorylate4E- selectively treat RAS-mutated cancer [174]. Lastly, treat- BP1 at the same residues (T37/46) that are phosphory- ment with everolimus or AZD8055 increases epidermal lated by mTOR and CDK1 [160]. Given that mTORC2 growth factor receptor (EGFR) activation in tumor cells, positively regulates Akt, the negative regulator of GSK3β, leading to drug resistance [175]. mTOR kinase inhibitor may paradoxically activate GSK3. Although PIK3CA-mutated cancer is usually sensitive to Hence, combinatorial inhibition of mTOR and GSK3β mTOR inhibition, activation of GSK3β in response to may synergistically suppress tumorigenesis. PI3K/mTOR inhibition may lead to the resistance to PI3K/mTOR inhibitors in PIK3CA-mutated cancer [176]. mTOR mutations A recent study demonstrates that lung squamous cell Gene mutations may affect the sensitivity of a drug that carcinoma adapt to chronic mTOR inhibition through the targets the protein encoded by this gene. More than 30 GSK3α/β signaling pathway, which involves the metabolic activating mutations of mTOR have been reported in reprogramming via increased glutaminolysis [177]. One human cancer, such as L1460P, C1483F, E1799K, F1888L, study also reveals that glutaminase (GLS) and glutamate T1977R, V2006I, V2046A, S2215Y, L2230V, E2388Q, levels are elevated in glioblastoma after treating with I2500F, R2505P, and D2512H [127, 161]. Cancer cells that mTOR inhibitor [178]. Treatment with GSK3 inhibitors harbor a subset of those mutations, including C1483F, or the glutaminase inhibitor effectively overcomes the E1799K, and S2215Y, are hypersensitive to rapamycin, resistance to mTOR inhibition [176–178]. Moreover, the whereas three mutations (A2034V, F2018L, and S2035F) activation of the purine salvage pathway due to increased in the FRB domain of mTOR are associated with rapa- expression of hypoxanthine phosphoribosyl transferase 1 mycin resistance [162, 163]. While tumor cells with muta- leads to the resistance to the dual PI3K/mTOR inhibitor tions in the kinase domain are still responsive to rapalogs gedatolisib [179]. In fact, mTOR is tightly involved in [161], mutations in the kinase domain of mTOR, such as purine metabolism. mTORC1 is not only activated by M2327I, S2215Y, L2230V, E2388Q, and V2046A, may be purine nucleobases or nucleosides [49], but also promotes responsible for the resistance to the ATP-competitive purine synthesis by ATF4-mediated upregulation of the inhibitor MLN0128 [127]. It remains to know whether mitochondrial tetrahydrofolate (mTHF) cycle enzyme activating mutations in the kinase domain of mTOR are methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) responsible for the resistance to allosteric mTOR kinase [180]. Moreover, mTORC1 promotes de novo pyrimi- inhibitors other than MLN0128. In addition, there are dine biosynthesis by S6K1-mediated phosphorylation recurrent mutations in other mTOR pathway genes, such of carbamoyl-phosphate synthetase 2, aspartate trans- as raptor, rictor,and RHEB [163]. RHEB-Y35N mutant carbamylase, and dihydroorotase (CAD) [181, 182]. gains the function to activate mTORC1 [161]. It warrants Therefore, the increased expression of hypoxanthine further studies to clarify which cancer-associated muta- phosphoribosyl transferase 1 may rescue the defect in tions in raptor, rictor, and RHEB may be associated with purine synthesis due to mTOR inhibition and help cancer mTOR inhibitors resistance. cells adapt to mTOR inhibition. Another compensatory response to mTORC1 inhibition Rewiring of oncogenic or metabolic pathways is the upregulation of transglutaminase 2, a multifunc- The sensitivity to mTOR inhibitors is regulated by other tional enzyme that is involved in cross-linking polypeptide oncogenic pathways, such as PI3K, MAPK, AURKA, and chains with e-(c-glutamyl)-lysine, apoptosis, signal trans- NF-kB signaling [164, 165]. Both the Ras/MAPK and duction, cell migration, cell adhesion, and extracellular Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 10 of 19 matrix remodeling [183–185]. Inhibition of transglutami- (PFS) among patients with progressive advanced pancreatic nase 2 potently sensitizes mTORC1-hyperactive cancer neuroendocrine tumors [197]. As registered in clinical- cells to rapamycin in vitro and in vivo [183]. Moreover, trials.gov,there aremorethan80clinicaltrialsfor mTOR mitochondria homeostasis is critical for cell growth and inhibitor monotherapy in cancer patients. A phase 2 trial survival. Mitochondrial hyperfusion is an adaptive of everolimus in patients with recurrent adult low-grade response to mTOR inhibition. Mechanistically, the gliomas demonstrates a high degree of disease stability translation of mitochondrial fission process 1 (MTFP1) [198]. Moreover, everolimus has a promising effect in is suppressed by mTOR inhibitors, which eventually patients with heavily pretreated, relapsed, or refractory results in mitochondrial hyperfusion, a process that classical Hodgkin’s lymphoma, with an overall response antagonizes apoptosis [186]. rate (ORR) of 45.6%, a median PFS of 8 months, and a long-term response (≥ 12 months) rate of 12% [188]. Of Clinical testing of mTOR inhibitors note, everolimus exhibits clinical activity as the first-line Given that preclinical studies demonstrate the anti-cancer monotherapy in a phase 2 clinical trial in 27 patients with efficacy of mTOR inhibitors alone or in combination with advanced biliary tract cancer [199]. Another phase 2 clin- chemotherapy, radiotherapy, and targeted therapy, there ical trial in 35 patients with thyroid cancer demonstrates are many completed or ongoing clinical trials to test the that everolimus has clinical benefit in patients with efficacy of mTOR inhibitors for treating various types of advanced differentiated thyroid cancer [200]. Also, single- human cancer (Table 1). In general, most of mTOR in- agent ridaforolimus has anti-tumor activity and acceptable hibitors are well tolerated, while there are some common tolerability in advanced endometrial cancer patients [201]. adverse effects including fatigue, rash, mucositis, and These observations need to be validated in a large scale of metabolic complications. mTOR inhibitors are associated randomized clinical trials. with a significantly increased risk of hyperglycemia, Based on a phase 2 trial in 167 patients, oral administra- hypertriglyceridemia, and hypercholesterolemia [187]. tion of the mTOR kinase inhibitor voxtalisib (50 mg, twice Other adverse events of everolimus are thrombocytopenia, daily) exhibits a promising efficacy in patients with folli- anemia, nausea, and stomatitis [188]. Ridaforolimus is cular lymphoma but limited efficacy in patients with orally bioavailable and better tolerated in children than mantle cell lymphoma, diffuse large B cell lymphoma, or the adults [189]. Deforolimus was well tolerated and chronic lymphocytic leukemia/small lymphocytic lym- showed encouraging anti-tumor activity across a broad phoma [202]. Of note, serious adverse events occurred in range of malignancies when administered intravenously, 58.1% of patients [202]. In contrast, the clinical efficacy of and a dose of 12.5 mg/day is being evaluated in phase II MLN0128 in patients with metastatic castration-resistant trials [190]. prostate cancer is limited, possibly due to the dose re- Moreover, MLN0028-treated patients may suffer from ductions secondary to toxicity [191]. Although it is anorexia, dyspenea and macunopapular rash [191]. In expected that mTOR kinase inhibitor may have superior clinical trials of solid tumors, the PI3K/mTOR inhibitor efficacy than rapalogs, a randomized phase 2 trial in NVP-BEZ235 (twice daily) is poorly tolerated, which patients with metastatic clear cell renal cancer demon- leads to treatment discontinuation in some patients and strated that the PFS and OS of AZD2014 were less than limits its efficacy in treating cancer [192, 193]. Apitolisib that of everolimus [203]. While the PI3K/mTOR inhibitor (GDC-0980), another dual pan-PI3K/mTOR inhibitor, NVP-BEZ235 is poorly tolerated in cancer patients, a also has grade 3–4 adverse effects and is less effective clinical trial in patients with recurrent endometrial cancer than everolimus [194]. GSK2126458 (GSK458) plus demonstrated that weekly intravenous administration of trametinib has poor tolerability, due to skin and gastro- another P3K/mTOR inhibitor gedatolisib achieved mo- intestinal toxicities such as diarrhea [195]. Daily oral derate anti-cancer activity with tolerable toxicity [204]. administration of PF-04691502 (8 mg/day) has adverse events including fatigue, nausea, vomiting, hypergly- mTOR inhibitors in combination therapy cemia, and rash [196]. The occurrence of the above- While mTOR inhibitor monotherapy has efficacy in some mentioned adverse effects following treatment with type of cancer, preclinical studies demonstrate strong mTOR inhibitors may be due to the critical roles of rationales for combinatorial treatment with mTOR in- mTOR in metabolism and immunity. hibitors and other drugs. For example, inhibition of both Akt/mTOR and WNT/β-catenin pathways synergistically mTOR inhibitors monotherapy suppresses AML [205]. As registered in clinicaltrials.gov, Everolimus has been approved by the FDA for the treat- there are many clinical trials to test the efficacy of mTOR ment of advanced renal cell carcinoma, pancreatic inhibitors in combination with other molecular targeted neuroendocrine tumors, and advanced breast cancer [83]. or chemotherapeutic agents. For example, everolimus is Everolimus significantly improves progression-free survival combined with one or several chemotherapeutic agents, Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 11 of 19 Table 1 Clinical evaluation of mTOR inhibitors mTOR inhibitor Category Combination Cancer type Phase Response PFS (months) OS (months) Ref. or trial ID* Everolimus (RAD001) Rapalog None Thyroid cancer 2 No CR/PR; SD (> 24 weeks) 9 (95% CI 4–14) 18 (95% CI 7–29) 200 58% Everolimus Rapalog Letrozole Relapsed ER(+) high- 2 CR 0; PR 16%; SD 37% 3.9 (95% CI 2.8–11); 13; 6-month OS rate, 209 grade ovarian cancer 3-month rate, 47%; 84% 6-month rate, 32% Everolimus Rapalog Exemestrane ER(+) locally advanced 3 CBR 33.4% vs 18% (control; 6.93 (95% CI 6.44–8.05) 30.98 (95% CI 27.96–34.56) NCT00863655 or metastatic breast placebo plus exemestrane) vs 2.83 (95% CI 2.74–4.14) vs control 26.05 (95% cancer (placebo plus exemestrane) CI 22.57–33.08) Everolimus Rapalog None Advanced neuroendocine 3 Not available 11.04 (95% CI 8.41–13.86) vs 44.02 (95% CI 35.61–51.75) vs NCT00510068 tumor placebo 4.6 (95% placebo 37.68 (95% CI 3.06–5.49) CI 29.14–45.77) Everolimus Rapalog Rituximab Diffuse large B cell 2 ORR 38% (90% CI 21–56%); 2.9 (90% CI 1.8–3.8) 8.6 (90% CI 4.9–16.3) 212 lymphoma CR 3/24; PR 6/24 NCT00869999 MLN0128 ATP-competitive Paclitaxel and Advanced solid tumors 1 CR 0; PR 8/54; SD (> 6 months) Not available Not available 87 trastuzumab 6/54 NCT01351350 AZD2014 (Vistusertib) ATP-competitive None Metastatic clear cell renal 2 Response rate 4% for AZD1024, 1.8 vs 4.6 for everolimus 4.9 for AZD1024 203 cancer 13% for everolimus treatment Progressive disease 69% vs 13% for everolimus treatment Voxtalisib (SAR24540; ATP-competitive None Relapsed or refractory 2 CR 8/164 (4.9%); PR 22/164 1.9 for follicular lymphoma Not available 202 XL765) non-Hodgkin lymphoma or (13.4%); Overall progression-free NCT01403636 chronic lymphocytic SD 55/164 (33.5%); ORR 18.3% rate at 24 weeks, 38·6% lymphoma (40.3% for follicular lymphoma) (95% CI 30·9–46·3) Gedatolisib (PKI-587; ATP-competitive None Recurrent endometrial 2 CR 1/38 (3%); PR 5/38 (13%); 3.7 (95% CI 2–5.6) for Not available 204 PF05212384) cancer SD > 16 weeks, 24% (37% for stathmin-low cancer; 3 NCT01420081 stathmin-low cancer, 11% for (95% CI 1.87–5.7) for stathmin-high cancer) stathmin-high cancer CR complete response, CBR clinical benefit rate, ORR overall response rate, OS overall survival, PFS progression-free survival, PR partial response, SD stable disease. *, Registration number in ClinicalTrials.gov Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 12 of 19 such as taxol, cisplatin, carboplatin, oxaliplatin, irinotecan, humanized monoclonal antibody against IGF-1R, and temozolomide, and gemcitabine. temsirolimus was tested in a clinical setting. This com- The phase 3 BOLERO-2 trial in patients with ER- bination shows clinical activity in patients with sarcoma positive/HER2-negative advanced or metastatic breast and adrenocortical carcinoma [215, 216]. In addition, a cancer demonstrates that a combination of everolimus combination of everolimus (5 mg daily) and the multi- and the aromatase inhibitor exemestane significantly im- kinase inhibitor sorafenib (400 mg twice daily) exhibits proves PFS, while the OS is not improved [206, 207]. Ac- anti-tumor activity in previously untreated patients with cordingly, a combination of everolimus and exemestane metastatic renal cell carcinoma with tolerable toxicity has been approved as a guideline for treating ER-positive/ [217]. However, a combination of sorafenib and evero- HER2-negative advanced or metastatic breast cancer limus fails to achieve the target of 6 month PFS of 50% [208]. In a phase 2 clinical trial, a combination of everoli- or greater among patients with unresectable high-grade mus and the aromatase inhibitor letrozole achieved a 12- osteosarcoma progressing after standard treatment week PFS rate of 47% in patients with ER-positive relapsed [218]. For patients with recurrent glioblastoma, a com- high-grade ovarian cancer [209]. In addition, the combin- bination of sorafenib (200 mg twice daily) and temsiroli- ation of everolimus with trastuzumab and paclitaxel has a mus (20 mg weekly) is associated with considerable promising efficacy in patients with highly resistant HER2- toxicity and poor efficacy [219]. positive advanced breast cancer (Table 1). This combin- In patients with metastatic castration-resistant prostate ation is currently under investigation in the BOLERO-1 cancer, a combination of everolimus and the EGFR phase 3 trial [210]. Moreover, a combination of everolimus inhibitor gefitinib has no significant anti-tumor activity with carboplatin is efficacious in treating metastatic triple- [220]. According to a phase 2 trial, a combination of negative breast cancer, with a median PFS of 3 months sunitinib and everolimus as the first-line therapy exhibits (95% CI 1.6 to 4.6 months) and overall survival (OS) of poor efficacy in treating advanced renal cell carcinoma 16.6 months [211]. In contrast, a combination of everoli- [221]. However, another phase 2 trial in patients with mus with gemcitabine/cisplatin has no synergistic effect in metastatic renal carcinoma demonstrates that the first- patients with metastatic triple-negative breast cancer. line sunitinib treatment followed by everolimus achieves Hence, this combination still needs validation in more a longer OS than the first-line everolimus followed by patients. sunitinib, suggesting that the sequence may affect the The CD20-targeted monoclonal antibody rituximab is a outcome [222]. Moreover, a combination of imatinib and treatment for low-grade or follicular CD20-positive non- everolimus has limited activity in the treatment of pa- Hodgkin’s lymphoma. Diffuse large B cell lymphoma tients with advanced chordoma [223]. The combination (DLBCL) is the most common type of non-Hodgkin’s of pimasertib and voxtalisib showed a poor long-term lymphoma. A phase 2 study of everolimus (10 mg/day) in tolerability and limited anti-tumor activity in patients combination with rituximab demonstrated an overall with advanced solid tumors [224]. response rate of 38%, a complete response rate of 12.5%, and a partial response rate of 25% among 24 patients with heavily pretreated DLBCL [212]. In addition, the combi- Concluding remarks nation of everolimus with rituximab or rituximab plus The discovery of TOR in yeast and mTOR in mammals cyclophosphamide, doxorubicin, vincristine, and pred- is a fundamental breakthrough in understanding cell and nisone (R-CHOP) was well tolerated in DLBCL patients organism growth, metabolism, and diseases. In-depth [212, 213]. It warrants further study to determine if the studies to clarify the regulators and effectors of mTOR combination of everolimus with R-CHOP has a better signaling have revealed multiple networks that work to- response in patients with DLBCL. In addition, the gether to integrate growth factors, nutrients, sterols, and combination of mTORC1/2 inhibitor with other targeted nucleotides signaling. The identification of the critical cancer drugs has been tested in clinical trials. Among 54 roles of mTOR and its regulators in tumorigenesis has cancer patients who were treated with MLN0128 and driven the development of the ever-growing list of trastuzumab/paclitaxel, 14.8% (8/54) of them achieved a mTOR inhibitors. While some of the mTOR inhibitors partial response, and near 11% (6/54) cases had stable have been approved to treat cancer patients, more disease for more than 6 months [87]. According to a phase mTOR inhibitors are under check to fulfill their promise 1 trial (NCT02193633), the combination of paclitaxel and for cancer therapy. vistusertib is highly active and well tolerated in patients It appears that mTOR inhibitors have mixed efficacy with high-grade serous ovarian cancer and squamous in patients with distinct kinds of cancer and among non-small cell lung cancer [214]. patients with the same kind of cancer. Recent studies Given that IGF-IR signaling may induce mTORC1 reveal that tumor organoids may help drug testing inhibitor resistance, the combination of cixutumumab, a [225, 226]. Tumor organoids may be used to test the Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 13 of 19 response of a given tumor to mTOR inhibitors. Alter- lethal with SEC13 protein 8; mSIN1: Mammalian stress-activated protein kinase-interacting protein 1; mTOR: Mechanistic target of rapamycin; natively, patient-derived tumor grafts may be transplanted PI3K: Phosphoinositide 3-kinase; PKC: Protein kinase C; PRAS40: 40 kDa to animals, followed by testing their response to mTOR proline-rich Akt substrate; Raptor: Regulatory-associated protein of mTOR; inhibitors [227]. It would be of interest to determine if these RCC: Renal cell carcinoma; RHEB: Ras homolog enriched in the brain; Rictor: Rapamycin-insensitive companion of mTOR; S6K1: Ribosomal protein emerging technologies are clinically relevant. S6 kinase β-1; TSC: Tuberous sclerosis complex In the era of precise medicine, it needs to determine if there are predictive biomarkers that may guide the Acknowledgements We would like to thank Qiulin Tang for her assistance in preparing the stratification of patients in clinical trials or help identify manuscript. the patients who most likely benefit from treatment with mTOR inhibitors in a clinical setting. Gene testing is a Authors’ contributions promising approach to achieve this goal. The candidates HH and YJ conceived the review and wrote the manuscript. QK and JW prepared the figures. HZ edited the references. TL was involved in editing for gene testing may include mTOR, PIK3CA, GATOR, the manuscript. All authors read and approved the final manuscript. KRAS, and BRAF. Mutations in PIK3CA and GATOR have been associated with higher sensitivity to mTOR Funding inhibition in preclinical studies. Hence, PIK3CA muta- This work was supported by grants 81672814 and 81872388 from the National Natural Science Foundation of China and grant 2018SCUH0009 tions may be potential sensitive markers. In contrast, from the Fundamental Research Fund for the Central Universities. KRAS/BRAF mutations may be resistant biomarkers. Both DNA from tumor samples and ctDNA from the Availability of data and materials Not applicable. blood may be subject to testing of gene mutations. In addition, gene mutations in the tumors may be dynamic Ethics approval and consent to participate during cancer evolution or regression [228]. It remains Not applicable. to determine if dynamic testing of ctDNA during the Consent for publication course of therapy may monitor cancer evolution and Not applicable. better predict drug resistance, thereby adjusting the treatment regimen in time. Recent progress in liquid Competing interests biopsy may help address this critical issue [229, 230]. In The authors declare that they have no competing interests. addition to gene testing, the solvable factors in the Author details blood may be potential biomarkers as well. Of particu- State Key Laboratory of Biotherapy, Laboratory of Stem Cell Biology, lar note, the mechanisms underlying the varied res- National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu 610041, China. Laboratory of Oncogene, Cancer Center, ponsiveness to mTOR inhibitors in cancer patients may West China Hospital, Sichuan University, Chengdu, China. School of Basic be complex. Rather than a single or few biomarkers, a Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, set of biomarkers may be more powerful and accurate China. Cancer Center, West China Hospital, Sichuan University, Chengdu, China. to meet the challenge. 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Medicine & Public Health; Oncology; Hematology; Cancer Research
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10.1186/s13045-019-0754-1
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Abstract

Mechanistic target of rapamycin (mTOR) is a protein kinase regulating cell growth, survival, metabolism, and immunity. mTOR is usually assembled into several complexes such as mTOR complex 1/2 (mTORC1/2). In cooperation with raptor, rictor, LST8, and mSin1, key components in mTORC1 or mTORC2, mTOR catalyzes the phosphorylation of multiple targetssuchasribosomal proteinS6kinase β-1 (S6K1), eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), Akt, protein kinase C (PKC), and type-I insulin-like growth factor receptor (IGF-IR), thereby regulating protein synthesis, nutrients metabolism, growth factor signaling, cell growth, and migration. Activation of mTOR promotes tumor growth and metastasis. Many mTOR inhibitors have been developed to treat cancer. While some of the mTOR inhibitors have been approved to treat human cancer, more mTOR inhibitors are being evaluated in clinical trials. Here, we update recent advances in exploring mTOR signaling and the development of mTOR inhibitors for cancer therapy. In addition, we discuss the mechanisms underlying the resistance to mTOR inhibitors in cancer cells. Keywords: Cancer, Drug resistance, mTOR, Oncogene, Targeted therapy Introduction mTORC3, consists of ETV7, mTOR, and other undefined The mechanistic target of rapamycin (mTOR) is a dual- components [3]. mTORC1 senses nutrients, growth factors, specificity protein kinase phosphorylating serine/threo- and cellular energy to orchestrate nucleotide, lipid, and nine as well as tyrosine residues [1]. Since the catalytic protein synthesis; inhibit autophagy; and stimulate cell domain of mTOR resembles that of lipid kinases such as growth [2]. mTORC2 is not only regulated by growth phosphoinositide 3-kinase (PI3K), mTOR is considered as factors, but also activates type I insulin-like growth factor an atypical protein kinase belonging to the PI3K-related receptor (IGF-IR) and insulin receptor (InsR) through the kinase family [2]. As a core component of several distinct tyrosine kinase activity of mTOR [1]. Besides, mTORC2 complexes including mTOR complex 1 (mTORC1), regulates the actin polarization and endocytosis [4, 5]. mTOR complex 2 (mTORC2), and a putative mTOR The mTOR signaling pathway has critical roles in complex 3 (mTORC3), mTOR has critical roles in diverse mammalian metabolism and physiology. The de-regulated biological processes, such as cell proliferation, survival, activity of mTOR is involved in many pathophysiological autophagy, metabolism, and immunity [2, 3]. While conditions, such as aging, Alzheimer’s disease, diabetes, mTOR and mammalian lethal with SEC13 protein 8 obesity, and cancer [2]. As a natural inhibitor of (mLST8) are common members of both mTORC1 and mTORC1, rapamycin is able to increase lifespan in mice mTORC2, regulatory-associated protein of mTOR (raptor), [6, 7]. mTOR activity is frequently de-regulated in a the 40 kDa proline-rich Akt substrate (PRAS40), and DEP variety of human cancers, such as breast, prostate, lung, domain-containing protein 6 (DEPTOR) are specific liver, and renal carcinomas. Upregulation of mTOR signa- members of mTORC1 [1, 2]. Instead, rapamycin-insensitive ling can promote tumor growth and progression through companion of mTOR (rictor) and mammalian stress- diverse mechanisms including the promotion of growth activated protein kinase-interactingprotein1(mSIN1or factor receptor signaling, angiogenesis, glyolytic meta- MAPKAP1) are unique components in mTORC2 but not bolism, lipid metabolism, cancer cell migration, and mTORC1 [1]. Another rapamycin-insensitive complex, suppression of autophagy [1, 2]. Hence, mTOR is a promising target for cancer therapy. In this review, we discuss the roles of mTOR in human cancer and * Correspondence: jyangfu@scu.edu.cn the rationales and challenges for developing mTOR Laboratory of Oncogene, Cancer Center, West China Hospital, Sichuan inhibitors to treat cancer. University, Chengdu, China Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 2 of 19 The assembly of mTOR complexes may stabilize the dimer by binding the HEAT repeats 11– The studies of mTORC1 structure demonstrate that 13 in one mTOR and repeats 20–22 in another mTOR [8, mTORC1 adopts a dimeric architecture with an overall 11]. In addition, raptor is required for recruiting substrates size of (280~300) × (200~210) × (100~130) Å [8, 9]. to mTORC1 [12, 13]. Both mTOR and raptor are sub- mTOR and LST8 form the core of mTOR complex that jected to phosphorylation at multiple residues (Fig. 1a), contains raptor and other regulatory proteins [8]. The which positively or negatively regulates mTORC1 activity. human mTOR contains 2549 amino acids that form TheassemblyofmTORC2and Saccharomyces cerevisiae several domains including the NH -terminal HEAT TORC2 follows a similar principle to mTORC1. The (N-HEAT), middle HEAT (M-HEAT), FAT, and kinase human mTORC2 structure reveals a hollow rhombohedral domain with a FRB insertion (Fig. 1). Raptor also fold with overall dimensions of ~ 220 × 200 × 130 (Å ) contains a HEAT domain, as well as WD40 and cas- [14]. A dimer of mTOR is located in the core of this pase-like domain [8, 9]. Besides, LST8 has WD40 domain. complex, while each mTOR or TOR heterodimerizes The HEAT motifs have conserved Asp and Arg residues with rictor and mSIN1 [14, 15]. Rictor has an NH -ter- at positions 19 and 25, respectively. A signature motif of minal armadillo (ARM) repeat cluster (~ 900 residues), WD40 repeats is ~ 40 amino acids often ending with a and the rest of the rictor is largely unstructured (Fig. 1b) tryptophan-aspartic acid (W-D) dipeptide [10]. The [16]. Interestingly, ARM and HEAT domains have similar HEAT repeats 12–13 in one mTOR interact with the conserved residues that form the hydrophobic domain HEAT repeats 20–23 in the M-HEAT domain of an- core and may have a common phylogenetic origin [17]. In other mTOR, thereby forming a dimer [8]. Raptor addition, mSin1 has a CRIM, a Ras-binding domain Fig. 1 The domains in key components of mTORC1 and mTORC2. a The molecular weight, domains, and phosphorylation sites in key components of mTORC1, including mTOR, LST8, and raptor. b The molecular weight, domains, and phosphorylation sites in key components of mTORC2, including mTOR, mSin1, and rictor Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 3 of 19 (RBD), and a pleckstrin homology (PH) domain [18]. an increase in AMP/ATP ratio, which activates the energy During the assembly of mTORC2, the FRB domain of sensor AMP-dependent kinase (AMPK). AMPK stimu- mTOR binds to mSin1 and the carboxy terminal region of lates the GAP activity of TSC and then promotes the rictor, while the NH -terminal portion (residues 506–516) inhibition of RHEB by TSC, leading to the downregulation of rictor interacts with the COOH-terminal region (resi- of mTORC1 [28]. In addition, the TCA cycle metabolite dues 1186-1218) of M-HEAT of mTOR [14]. In addition, ketoglutarate inhibits mTORC1 through repressing ATP mSin1 directly binds to rictor. Both rictor and mSin1 are synthase, increasing AMP/ATP ratio and activating responsible for recruiting substrates to mTORC2. Of note, AMPK [29]. Cellular energy deficiency usually leads to both rictor and mSin1 have mTOR-independent partners. endoplasmic reticulum stress, which in turn induces the For example, rictor interacts with integrin-linked kinase unfolded protein response (UPR). Ire1, ATF6, and PERK and promotes its phosphorylation of Akt [19], while are three major mediators of the UPR. Upon ER stress, mSin1 interacts with Ras and inhibits ERK1/2 phosphory- ATF6 can induce RHEB expression, which in turn lation [20]. Thus, the outcome from the manipulation of promotes mTORC1 activation and cell survival [30]. rictor or mSin1 alone may not exactly reflect the function However, overactivated mTORC1 is also harmful to of mTORC2. cell survival under ER stress. Mutations in TSC1/2 or activation of RHEB renders cells hypersensitive to ER Regulation of mTORC1 activity stress-induced apoptosis, which may be due to the The activity of mTORC1 is regulated by growth factors, downregulation of ATF4/6 by mTOR [31]. Therefore, cellular energy, stresses and nucleotides, etc. The lyso- mTORC1 may have versatile effects on cell survival somes are primary sites for mTORC1 activation. The under ER stress. activation of mTORC1 by growth factors is dependent on While the regulation of mTORC1 by growth factors is Ras homolog enriched in the brain (RHEB), a lysosomal dependent on RHEB and the TSC complex, amino acids GTPase that directly interacts with mTOR and activates it can stimulate mTORC1 independent of TSC. The regula- [21]. Upon binding to growth factors such as epidermal tion of mTORC1 by amino acids is very complicated, growth factor (EGF) and insulin-like growth factor (IGF), involving multiple amino acid sensors and protein the growth factor receptors (EGFR, IGFR, etc.) are machinery [32]. The lysosomal Ragulator (RAG) guano- activated, which in turn activate PI3K-PDK1-Akt signaling sine triphosphatases (GTPases) play key roles in the pathway. Active Akt phosphorylates tuberous sclerosis activation of mTORC1 by amino acids. RAGA or RAGB complex 2 (TSC2) and inhibits the TSC complex, a heterodimerizes with RAGC or RAGD [33]. Further, RAG GTPase-activating protein (GAP) complex consisting of proteins form a large complex with LAMTOR1/2/3/4/5, TSC1/2 and TRE2-BUB2-CDC16 domain family member which recruit RAG and mTORC1 to the lysosomal surface 7 (TBC1D7) [22, 23]. The TSC complex can inactivate [34]. The activity of RAG is regulated by two complexes, RHEB thereby inhibiting mTOR [24]. Therefore, the GATOR1 and GATOR2. GATOR1, which is composed of activation of Akt leads to the depression of RHEB and DEPDC5, NPRL2, and NPRL3, inhibits the GTPase- then activates mTORC1. Moreover, the ubiquitination of activated protein (GAP) activity of RAGA/B thereby RHEB regulates its ability to activate mTORC1 [21]. The repressing the activation of mTORC1 by amino acids [35]. E3 ubiquitin ligase RNF152 catalyzes RHEB ubiquitina- Instead, GATOR2, a protein complex consisting of tion, leading to an increase in the interaction between MIOS, WDR24, WDR59 SEH1L, and SECB, negatively RHEB and TSC [21]. In contrast, Akt can phosphorylate regulates GATOR1 by inducing DEPDC5 degradation the deubiquitinase USP4 that promotes RHEB deubiquiti- [35]. Furthermore, KICSTOR, a large complex consisting of nation thereby releasing RHEB from TSC [21]. KPTN, ITFG2, C12ORF66, and seizure threshold 2 (SZT2), Downstream of the growth factor receptors, the recruits GATOR1 to the lysosomal surface and mediates mitogen-activated protein kinase (MAPK) also up- the interaction between GATOR1 and RAG [36, 37]. regulates mTORC1 activity. Mechanistically, MEK1/2 Sestrin (SESN) is another category of negative inhibitors promotes raptor phosphorylation through ERK1/2 and of amino acid-induced mTORC1 activation. Mechanis- p90 ribosomal S6 kinase (RSK1/2). ERK1/2 directly tically, SESNs interact with GATOR2, leading to the phosphorylates raptor at S8, S696, and S863, while release of GATOR1 from GATOR2. The released RSK1/2 phosphorylates raptor at S719/722 [25, 26]. GATOR1 in turn inhibits RAG and mTORC1 [38–40]. Of Meanwhile, the intestinal cell kinase (ICK), a MAPK- note, SESN2 is known as a leucine sensor in mTORC1 related kinase, phosphorylates raptor at T908 [27]. signaling. Leucine directly binds to SESN2, leading to Phosphorylation of raptor by ERK/RSK/ICK promotes the dissociation of SESN2 from GATOR2. The re- the activation of mTORC1. leased GATOR2 binds to GATOR1 and then prevents the mTORC1 not only senses growth factors, but also inhibition of RAG by GATOR1. These sequential pro- responds to cellular energy. Low cellular energy results in cesses result in RAG-mediated mTORC1 activation [41]. Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 4 of 19 To prevent the overactivation of mTORC1 by amino How does mTORC2 respond to cellular energy and nu- acids, there are negative feedback pathways to RAG- trients? The energy sensor AMPK inhibits mTORC1 and mediated mTORC1 activation. Two E3 ubiquitin then releases the suppression of mTORC2 by mTORC1, ligases, RNF152 and SKP2, reportedly induce RAGA leading to the activation of mTORC2 [56]. Thus, upregu- ubiquitination and potentiate the binding of RAGA to lation of mTORC2 may help cells adapt to low levels of GATOR1 [42, 43]. While leucine sufficiency is sensed cellular energy. Moreover, mTORC2 is activated by by SESN2, the stimulation of mTORC1 by arginine is glutamine starvation. Activated mTORC2 upregulates the mediated by SLC38A9 [44]. Moreover, the ubiquitin expression and phosphorylation of glutamine:fructose-6- ligase TRAF6 can catalyze K63 ubiquitination of both phosphate amidotransferase 1 (GFAT1), the rate-limiting Akt and mTOR thereby promoting the activation of enzyme of the hexosamine biosynthesis pathway (HBP) Akt and mTORC1 by amino acids [45, 46]. [57, 58]. A study of budding yeast demonstrates that the In addition, mTOR may be activated by lipid and LKB1-ELM1-GIN4/HSL1 axis is required for coordinating cholesterol. Fatty acid metabolism leads to the de novo TORC2 signaling to the changes in carbon source [59]. It synthesis of phosphatidic acid (PA), which stabilizes both remains to know if similar pathway works in human mTORC1 and mTORC2 [47]. Moreover, cholesterol can cancer cells. stimulate mTORC1 activation and growth signaling. Similar to mTORC1, mTORC2 is also stabilized by Mechanistically, SLC38A9 acts as a lysosomal choles- phosphatidic acid (PA), a central metabolite in the synthe- terol sensor to stimulate the activation of mTORC1 by sis of membrane phospholipids [60]. The generation of PA RAG complex [48]. Recently, it was reported that is catalyzed by the phospholipase D, diacylglycerol kinases, mTORC1 is also responsive to the levels of purine and lysophosphatidic acid acyltransferases. Moreover, the nucleotides [49]. While adenylate stimulates mTORC1 activity of mTORC1 and mTORC2 is regulated by mLST8 by inhibiting TSC, guanylate downregulates RHEB and ubiquitination. It has been reported that the E3 ubiquitin then inhibits mTORC1 [49]. The mechanisms under- ligase TRAF2 positively regulates K63-linked polyubiquiti- lying the regulation of TSC and RHEB by adenylate and nation of mLST8, which impairs its interaction with guanylate remain to be known. mSin1 and compromises the mTORC2 integrity, but enhances the assembly of mTORC1 [61]. On the contrary, Regulation of mTORC2 activity the deubiquitinase OTUDB7 removes polyubiquitin Although mTORC1 and mTORC2 are distinct com- chains from G L to promote G L interaction with mSin1 β β plexes, there is a crosstalk between these two complexes. and the integrity of mTORC2 [61]. Besides, the exchange On one hand, mTORC2 can activate IGF-IR-Akt axis factor found in platelets, leukemic, and neuronal tis- thereby upregulating mTORC1 [1]. On the other hand, sues (XPLN) interacts with mTORC2 and negatively mTORC1 feeds back to inhibit mTORC2 via S6K1, one regulates mTORC2 activity [62]. Lastly, mTOR is a of the substrates of mTORC1. Once activated by target of proteasomal degradation when it is ubiquiti- mTORC1, S6K1 phosphorylates rictor and mSin1 on nated by FBXW7 [63]. T1135 and T86/398, respectively, leading to the impair- ment of mTORC2 integrity [50–52]. Targets of mTORC1 and mTORC2 While mTORC2 directly activates IGF-IR and InsR, As a protein kinase, mTOR catalyzes the phosphorylation receptor tyrosine kinases such as EGFR, PDGFR, and of its targets and regulates their activity. mTORC1 and IGF-IR can activate mTORC2 via PI3K. Mechanistically, mTORC2 have different substrates. While the repertoire PI3K-induced PtdIns (3,4,5) P3 (PIP3) binds to the PH of mTOR substrates keeps increasing, there are more domain of mSin1 and then disables the inhibition of targets remaining to be identified. S6K1 and 4E-BP1 are mTOR kinase domain by mSin1, thereby activating two well-known mTORC1 targets. mTORC1 phosphory- mTORC2 [18]. In addition, PI3K promotes the asso- lates S6K1 at T389 and 4E-BP1 at multiple residues [64]. ciation of mTORC2 with ribosome, where mTORC2 is Phosphorylation of S6K1 by mTORC1 leads to increased activated [53]. Therefore, mTORC2 also responds to protein and nucleotide synthesis. While 4E-BP1 is a nega- growth factors. Notably, another study suggests that tive regulator of 5′cap-dependent mRNA translation, mTORC2 activity is localized in the plasma membrane, phosphorylation of 4E-BP1 by mTORC1 induces its mitochondria, and endosomal vesicles, and the activity dissociation from eIF4E, thereby relieving its inhibition of of mTORC2 via the mSin1-PH domain at the plasma protein synthesis [65]. To cope with increased protein membrane is PI3K- and growth factor-independent [54]. synthesis, mTORC1 also promote ribosome biogenesis by In addition, IKKα interacts with mTORC2 and enhances inducing ribosomal RNA transcription. Mechanistically, its kinase activity towards Akt [55]. These data suggest mTORC1 may translocate to the nucleus, where it binds that the activation of mTORC2 involves multiple to ribosomal DNA promoter [66–68]. Nuclear mTOR also location and different mechanisms. phosphorylates TFIIIC and Maf1, thereby promoting Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 5 of 19 tRNA gene transcription [69]. In fact, nuclear mTOR reg- various kinds of cancer, such as breast cancer, gastric can- ulates RNA polymerase 1/2/3-driven transcription. In cer, and liver cancer [78, 79]. Moreover, rictor is overex- addition, mTORC1 phosphorylates the E3 ubiquitin ligase pressed in human cancers of the brain, breast, lung, gastric, SKP2 at S64 and then inhibits SKP2 ubiquitination and colon, liver, and tongue [80, 81]. degradation [70]. Given that SKP2 promotes the de- Given that mTOR has critical roles in tumor progres- gradation of many proteins, mTORC1 may regulate the sion, mTOR inhibitors hold promise in cancer therapy. turnover of SKP2 substrates indirectly. Thus, mTORC1 Indeed, rapamycin analogs (rapalog) have been approved not only promotes protein synthesis, but also regulates for treating cancer in the clinic. In addition, many protein degradation. mTOR inhibitors with different mechanisms of action Following the identification of mTORC2, it was found have been developed, some of which are undergoing that protein kinase C (PKC) α/β were the substrates of clinical trials in variety types of human cancer. mTORC2 that regulates the actin cytoskeleton [4, 71]. Moreover, mTORC2 phosphorylates and activates other Rapalog AGC kinases, such as serum and glucocorticoid-induced Rapamycin was originally identified as an antifungal, kinase (SGK) and Akt. mTORC2 phosphorylates Akt at immunosuppressive, and antiproliferative agent. Later S473, leading to allosteric activation of Akt in cooperation studies revealed that rapamycin binds to the 12 kDa with the catalytic activation by PDK1, which phosphory- FK506-binding protein (FKBP12) and then inhibits lates Akt at T308 [72]. During the synthesis of nascent mTORC1 [82]. Since rapamycin has poor solubility proteins, mTORC2 can co-translationally phosphorylate and pharmacokinetics, it is not suitable for treating some polypeptides while they are attached to the ribo- human cancer. So far, several water-soluble rapamycin some. IGF2 mRNA-binding protein (IMP) is responsible analogs have been developed. For example, temsiroli- for the splicing and translation of IGF2 mRNA. mTORC2 mus and everolimus exhibit tumor-suppressive effects co-translationally phosphorylates IMP1 at S181 and then in vivo. Both temsirolimus and everolimus have been promotes IMP1 binding to the untranslated region of used to treat advanced renal cell carcinoma (RCC) in IGF2 mRNA and enables translational initiation by the clinic. Moreover, everolimus is prescribed for internal ribosomal entry [73]. mTORC2 not only enhances treating pancreatic neuroendocrine tumors and advanced the production of IGF2 protein, but also phosphorylates breast cancer [83]. Besides, there are many clinical trials and activates IGF-IR and insulin receptor [1]. In contrast to evaluate the efficacy of rapalogs in treating other to mTORC1’s activity as a ser/thr kinase, mTORC2 has types of human cancer, such as advanced gastric tyrosine kinase activity towards IGF-IR/InsR [1]. cancer, hepatocellular carcinoma, non-small cell lung cancer, endometrial cancer, and mantle cell lymphoma mTOR inhibitors for cancer therapy (clinicaltrials.gov). The activity of mTOR is frequently upregulated in Of particular note, the effect of rapalog monotherapy human cancer. The aberrant activation of mTOR in human on solid tumors is modest in the clinic. The incomplete cancer may be attributed to mTOR pathway-activating inhibition of mTOR by rapalogs may result in limited mutations, amplification, or overexpression of the com- clinical success. On the other hand, inhibition of ponents of mTOR complexes and mutations or loss of mTORC1 may lead to feedback activation of IGF-IR and negative regulators of mTOR. PIK3CA mutations are Akt, which compromises the anti-cancer effect of frequently detected in human cancer. Activation of PI3K rapalogs [1]. Taking into account the complexity of promotes both mTORC1 and mTORC2 activation. In mTOR signaling networks, it is not hard to understand addition, mutations in KRAS and BRAF may lead to that the response to rapalogs varies in patients with mTORC1 activation. Especially, KRAS can directly bind to cancer, such as metastatic RCC. It is desirable that there PIK3CA (p110α) and activates PI3K pathway, leading to are biomarkers to predict the responses to mTOR in- mTOR activation [74]. mTOR-activating mutations are ob- hibition. KRAS, BRAF, and TSC mutations are known as served in kidney cancer. While mTOR activity is usually resistant markers for mTOR inhibitors, whereas PIK3CA upregulated by growth factors and amino acids, activating mutations are sensitive marker [84, 85]. However, the mutations in mTOR may result in RAG- and RHEB- roles of TSC1/2 and mTOR mutations in responding to independent mTOR hyperactivation, thus loss of the de- rapalogs remain controversial. Although it has been pendency on growth factors and amino acids [75]. Point reported that mutations in TSC1/2 and mTOR are more mutations in RHEB and GATOR1 were also detected in frequent in RCC patients who respond well to rapalogs, renal cancer and endometrial cancer [76]. RHEB1 is over- the majority of rapalog responders have no mutations in expressed in acute myeloid leukemia (AML) and promotes mTOR pathway, suggesting that other factors are also AML progression [77]. Whereas mTOR amplification is involved in rapalog sensitivity [86]. Notably, rapalogs rare in human cancer, rictor amplification is detected in usually arrest cell proliferation but does not induce Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 6 of 19 apoptosis. Despite the initial response, tumors frequently volume in cisplatin-resistant ovarian cancer model [104]. develop resistance to these agents. Similar to PP242, AZD2014 enhances the radiosensitivity of glioblastoma stem-like cells [105]. Based on the above- ATP-competitive mTOR inhibitors described studies, it appears that the pan-mTORC1/2 in- To more completely inhibit mTOR, a number of ATP- hibitors generally reverse rapalog resistance, endocrine re- competitive mTOR inhibitors have been developed to sistance, chemoresistance, and radioresistance. target both mTORC1 and mTORC2. Tumors that are addicted to the mTOR signaling pathway may be Dual PI3K/mTOR inhibitors sensitive to this kind of inhibitors. Unlike rapalogs, ATP- Although inhibition of mTORC1 and mTORC2 can competitive mTOR inhibitors can not only arrest cell downregulate Akt S473 phosphorylation, mTOR inhibi- growth, but also induce apoptosis. MLN0128 (also called tors may paradoxically enhance the PI3K/PDK1 axis. INK128, sapanisertib, TAK-228) is a pan-mTOR in- Thus, an inhibitor targeting both PI3K and mTOR may hibitor that has potent in vitro and in vivo anti-tumor have better anti-cancer activity compared to targeting effects, and has underwent clinical trials for solid tumors mTOR alone [106, 107]. Due to the similarity between such as bone and soft tissue sarcoma, breast cancer, and PI3K and mTOR, some chemicals can inhibit both PI3K primary effusion lymphoma, a non-Hodgkin B cell and mTOR. NVP-BEZ235 (dactolisib) inhibits the activity lymphoma that usually results from infection of Kaposi of multiple class I PI3K isoforms, mTOR and ataxia sarcoma-associated herpesvirus [87–90]. MLN0128 also telangiectasia, and Rad3-related protein (ATR) and has reduces tumor growth in CD44-high HCC xenografts potent anti-cancer activity [108]. Notably, NVP-BEZ235 and resensitizes HCC to sorafenib [91]. Of note, can penetrate the blood-brain barrier after systemic MLN0128 is an effective agent even in tumors that are administration [109]. Therefore, it can be used to treat resistant to rapamycin or chemotherapy. A recent study glioma and reverse temozolomide resistance [110]. In demonstrates that MLN0128 can overcome resistance to addition, NVP-BEZ235 can suppress paclitaxel-resistant everolimus and reduce tumor size by 20% in PIK3CA- gastric cancer, which exhibits increased PI3K/mTOR mutant colorectal cancers [92]. In addition, MLN0128 activity [111]. can induce tumor shrinkage in patient-derived xenograft LY3023414, a complex fused imidazoquinolinone, is model of pancreatic neuroendocrine tumors, even in an oral PI3K/mTOR and DNA-PK inhibitor that has everolimus-resistant tumors [93]. anti-tumor effects in animal models. Combination of PP242 (Tokinib) is another selective ATP-competitive LY3023414 with standard chemotherapeutic drugs has inhibitor of mTOR that has a promising anti-cancer additive anti-tumor activity [112, 113]. Another dual activity over several cancer types, such as leukemia, PI3K/mTOR inhibitor voxtalisib (SAR245409, XL765), a gastric cancer, and colon cancer [94, 95]. Given that pyridopyrimidinone derivative, significantly inhibits tumor the Akt-mTOR signaling pathway is upregulated in growth in multiple human xenograft models [114]. Com- platinum-resistant cancer cells, studies demonstrate bination of voxtalisib and the MEK inhibitor pimasertib that mTORC1/2 inhibitor, such as PP242 and MLN0128, synergistically inhibits certain endometrial cancer cells can re-sensitize platinum-resistant ovarian cancer cells to growth [115]. Other dual PI3K/mTOR inhibitors include carboplatin in vitro and in vivo [96, 97]. Mechanistically, PQR309, XH00230381967, SN20229799306, GSK2126458 mTOR inhibition leads to a sharp decrease in the transla- (omipalisib), and PKI-587. tion of DNA damage and repair response and pro-survival Of note, PQR309 is a 4,6-dimorpholino-1,3,5-triazine- mRNAs, including CHK1 [98]. Consistent with the in- based, brain-penetrant, and orally bioavailable PI3K/ hibition of DNA repair, mTOR inhibitors are also effective mTOR inhibitor [116]. PQR309 effectively inhibits lym- in enhancing radiosensitivity or restoring radiosensitivity phoma in monotherapy and in combination therapy in radioresistant tumors [99, 100]. Moreover, inhibition of with other drugs, such as the BCL2 inhibitor veneto- mTORC1/C2 signaling improves anti-leukemia efficacy of clax, the HDAC inhibitor panobinostat, the Bruton’s JAK/STAT blockade in CRLF2-rearranged and/or JAK- tyrosine kinase inhibitor ibrutinib, lenalidomide, the driven Philadelphia chromosome-like acute B cell lympho- BET proteolysis-targeting chimera ARV-825, the prote- blastic leukemia [101]. asome inhibitor marizomib, and the anti-CD20 mono- Both AZD2014 (vistusertib) and its analog AZD8055, clonal antibody rituximab [117]. Moreover, PQR309 can two ATP-competitive mTORC1/2 inhibitors, are highly suppress cancer cells with primary or secondary resistance effective in treating estrogen receptor (ER)-positive breast to the PI3Kδ. PQR620 and the PI3K/mTORC1/2 inhibitor cancer. Moreover, AZD2014 and AZD8055 can suppress PQR530 effectively cross the blood-brain barrier [118]. breast cancer with acquired resistance to endocrine The dual specificity PI3K/mTOR inhibitor gedatolisib therapy, rapalogs, and paclitaxel [102, 103]. In addition, a (PKI-587, PF05212384) is a bis(morpholino-1,3,5-tria- combination of AZD2014 with paclitaxel reduces tumor zine) derivative [119]. Gedatolisib inhibits tumor growth Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 7 of 19 in breast, colon, lung, and glioma xenograft models and Principle mechanisms of mTOR inhibitor resistance displays efficacy against T cell acute lymphoblastic in cancer leukemia (T-ALL) and Philadelphia chromosome (Ph)- Drug resistance is a serious problem in treating cancer. like B cell acute lymphoblastic leukemia (Ph-like ALL) Although there may be an initial response, long-lasting [107, 120]. Combination of gedatolisib with ruxolitinib treatment with chemotherapeutic or molecular-targeted or dasatinib has superior efficacy than a single agent in drugs often faces the challenge of drug resistance. Due to CRLF2/JAK-mutant models and ABL/PDGFR-mutant the tumor heterogeneity, some tumors do not respond to models, respectively [120]. In addition, gedatolisib a given drug at all. Clonal selection, adaptive evolution, sensitizes head, neck, and nasophageal carcinoma to ra- and resistance to cell death are general mechanisms for diation therapy [121, 122] and sensitizes EGFR-resistant drug resistance. Due to the complexity and crosstalk in head and neck carcinoma to cetuximab [123]. Thus, signaling networks, cancer cells may adapt to an inhibitor gedatolisib may be a candidate sensitizer to radiotherapy that targets a given signaling pathway via the com- and targeted therapy. pensatory activation of other pathways. Although mTOR GSK2126458 (omipalisib) is an orally bioavailable inhibitors exhibit potent anti-cancer effects in many inhibitor of PI3Kα and mTOR [124]. Omipalisib potently preclinical models, resistance does occur. As described inhibits FGFR4-V550E tumor-derived cell and human below, there are multiple mechanisms underlying the rhabdomyosarcoma cell viability and reduces the growth resistance to mTOR inhibitors (Fig. 2). of rhabdomyosarcoma in vivo [125]. In addition, a combination of the PI3K/mTOR inhibitor VS-5584 and Drug efflux by ATP binding cassette transporters the Wnt inhibitor ICG-001 synergistically inhibits AML ATP-binding cassette (ABC) transporters constitute drug with high PRL-3 expression [126]. Finally, the efficacy efflux pumps that decrease the intracellular levels of of mTOR inhibitor may be enhanced by linking the drugs, leading to poor treatment outcome. Overexpres- kinase inhibitor to rapamycin (RapaLink) [127]. EZH2 sion of ABC transporters is a general mechanism for (Y641X)-mutant lymphomas show increased sensiti- multi-drug resistance in cancer. The same may be true vity to RapaLink-1 [128]. Given that RapaLink in- for mTOR inhibitor resistance. In fact, the mTOR in- tegrates the activity of both rapamycin and mTOR hibitors rapamycin and NVP-BEZ235 are substrates of kinase inhibitor, it is worthwhile looking forward to ABCB1 (P-glycoprotein) and ABCG2 (also called breast the efficacy in clinical trials. Lastly, there are many cancer resistance protein, BCRP), respectively [132]. In drugs that may indirectly inhibit mTOR, such as addition, AZD8055 is transported by both ABCB1 and aspirinand metformin[129–131]. ABCG2 [132]. Fig. 2 The mechanisms for resistance to mTOR inhibitors in cancer cells. ABC transporters, ATP binding cassette transporters; EMT, epithelial-mesenchymal transition Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 8 of 19 Studies show that ABCB1 is overexpressed in luminal FGF1-FGFR-Notch1 axis [147]. Blocking FGFR or Notch1 breast cancer cell lines that are resistant to everolimus may prevent resistance to TORC1/2 inhibitors by abro- [133]. Also, ABCB1 inhibits brain accumulation of gating the expansion of drug-resistant CSCs in TNBC everolimus [134]. Overexpression of ABCG2 in cancer [49]. Moreover, another dual PI3K/mTOR inhibitor PF- cells confers significant resistance to PF-4989216, which 04691502 can induce a stem cell-like gene expression can be reversed by an inhibitor or competitive substrate signature in KRAS-mutant colorectal cancer models of ABCG2 [135]. Moreover, GDC-0980 is subject to [148]. Together, these data suggest that the effects of active efflux by ABCB1 and BCRP, which limits its mTOR inhibitors on CSC may be dependent on the genetic efficacy [136]. The affinity for ABC transporters may background and rewiring of cancer stemness pathways. vary among different mTOR inhibitors. Lowering the affinity for ABC transporters or inhibiting ABC trans- Assembly of the translation machinery porters may enhance the efficacy of mTOR inhibitors. Eukaryotic protein synthesis is regulated by several me- chanisms including cap-dependent and cap-independent Cancer stem cells translation. The cap-dependent pathway involves many Cancer stem cells (CSCs) are a subpopulation in tumor eukaryotic initiation factors (eIF), such as eIF1, eIF2, eIF3, mass that is extremely resistant to standard cancer therapy. eIF4A, eIF4B, eIF4E, eIF4H, eIF5, and eIF6. The protein Slow-cycling CSC is one of the major obstacles to eradicate synthesis is initiated by the association of the 40S ribo- tumor [137]. It is generally thought that the mTOR some subunit with eIF1A and eIF3, followed by binding of pathway is hyperactivated in CSC. Transforming growth the eIF2-GTP-methionine tRNA complex to 40S subunit factor-β (TGF-β) can induce epithelial-mesenchymal tran- and then forming a 43S subunit [149]. The eIF4F complex, sition (EMT), which enhances cancer stem cell generation. which consists of eIF4E, eIF4A, and eIF4G, binds to the mTOR is one of the mediators in TGF-β signaling path- m G cap at the 5′ end of mRNA and then activates ways that enhances cancer stemness and drug resistance mRNA. The activated mRNA is recruited to the 43S com- [138]. The inhibitory effect on CSCs has already been plex and then subjected to ATP-dependent scanning of shown for some mTOR inhibitors [139]. Rapamycin, mRNA to locate the initiating AUG code [150]. Finally, everolimus, and PF-04691502 suppress tamoxifen-induced the 60S ribosome subunit is associated with the 40S sub- activation of breast cancer stem cells [140]. Inhibition of unit to form the 80S initiation complex, possibly assisted mTOR restores tamoxifen resistance in breast cancer cells by eIF5. For the initiation of cap-independent protein [141]. Moreover, the ATP-competitive mTOR inhibitor synthesis, the 40S ribosome subunit binds to an internal Torin1 and PI3K/mTOR inhibitor VS-5584 preferentially region of mRNA, which is referred to as internal ribosome reduce CSC levels in multiple mouse xenograft models of entry sites (IRES), or the untranslated regions of mRNA. human cancer [142, 143]. Given that stimulation of cap-dependent translation is However, the interplay between mTOR inhibitors and one of the major functions of mTORC1, the status of the CSC is complex. Previous studies show that expansion translation machinery and modes of protein translation of CSC promotes the resistance to mTOR inhibitor in may impact on the efficacy of mTOR inhibitors. 4E-BPs leiomyosarcoma [144]. PDK1 signaling toward PLK1- are phosphorylated and inactivated by mTORC1. The MYC activation leads to tumor-initiating cell activation sensitivity to PP242 is correlated with the extent to which and resistance to mTOR inhibition [145]. Inhibition of 4E-BP1 phosphorylation is inhibited by this drug [151]. EZH2, a catalytic component of polycomb repressive Loss of 4E-BPs in tumor cells results in the resistance to complex which plays a critical role in stem cell main- mTOR inhibition. The transcription factor Snail directly tenance, restores sensitivity to PI3K/mTOR pathway represses 4E-BP1 transcription and compromises the anti- inhibition. It appears that the sensitivity to mTOR in- cancer effects of mTOR inhibitors [152]. Of note, Snail is hibitors in CSC may be context- or cell type-dependent. translationally regulated by eIF4E, which is exactly the Of note, one study demonstrates that TP53 mutation target of 4E-BP. Phosphorylation of eIF4E (S209, etc.) and BCL2 phosphorylation affect the sensitivity of promotes Snail synthesis [153]. Therefore, 4E-BP and glioblastoma stem-like cells to mTOR inhibitor [146]. eIF-4E can disable each other. Overexpression of BCL2 (T56/S70) phosphorylation in TP53 wild-type eIF4E or phosphorylation of eIF4E (S209) by MAP glioblastoma stem-like cells is responsible for the lower kinase-interacting kinase 1 (Mnk1/2) leads to a shift from sensitivity to the mTORC1/2 inhibitor AZD8055, as - cap-dependent to cap-independent translation and then compared to TP53-mutated glioblastoma stem-like cells renders cancer cells insensitive to mTOR inhibition [146]. In addition, while mTOR inhibitors reportedly sup- [154, 155]. Thus, inhibition of Mnk1/2 or its up- press CSC, one study demonstrates that treatment of stream kinase ERK1/2 may restore cap-dependent TNBC cell lines with PI3K/mTOR inhibitor or TORC1/2 translation and the sensitivity of mTOR inhibitors inhibitor expands CSC population through upregulating [155]. On the other hand, inhibition of mTORC1 may Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 9 of 19 lead to paradoxical phosphorylation of eIF4E in PI3K- PI3K/Akt/mTOR pathways are tightly involved in tumori- and Mnk-dependent manner and promote cap-independent genesis. While tumors with PIK3CA/PTEN mutations or translation [156]. Hence, a combination of mTOR Akt hyperactivation usually are sensitive to mTOR inhi- and Mnk inhibitors is an effective therapeutic strategy bitors, KRAS/BRAF mutations are predictive biomarkers for cancer [157]. of mTOR inhibitor resistance [148, 166–169]. In addition, Notably, 4E-BP1 is not only phosphorylated by mTORC1, mTOR inhibition may lead to the activation of the but also phosphorylated and inactivated by other kinases MEK-Erk pathway. Combination of RAF/MEK inhibi- such as CDK1, CDK12, and GSK3 . CDK1 can substitute tors and mTOR inhibitors may be a strategy to treat mTORC1 to phosphorylate 4E-BP1 and activate cap- KRAS-mutated cancer [170, 171]. Besides, the activation dependent translation, which is resistant to mTOR of Erk in response to mTOR inhibition can be abrogated inhibition [158]. In addition, CDK12 cooperates with by the CDK4/6 inhibitor palbociclib [172]. Combination mTORC1 to phosphorylate 4E-BP1 and releases it from of CDK4/6 and mTOR inhibitors synergistically inhibits mTORC1 target mRNAs thereby promoting their tumor growth [172, 173]. Alternatively, combined in- translation [159]. Therefore, combinatorial inhibition of hibition of wee1, a protein kinase that regulates the G2 mTOR and CDK1/12 may be synthetically lethal to cancer checkpoint in the cell cycle, with mTOR inhibition may cells. Furthermore, GSK3β can directly phosphorylate4E- selectively treat RAS-mutated cancer [174]. Lastly, treat- BP1 at the same residues (T37/46) that are phosphory- ment with everolimus or AZD8055 increases epidermal lated by mTOR and CDK1 [160]. Given that mTORC2 growth factor receptor (EGFR) activation in tumor cells, positively regulates Akt, the negative regulator of GSK3β, leading to drug resistance [175]. mTOR kinase inhibitor may paradoxically activate GSK3. Although PIK3CA-mutated cancer is usually sensitive to Hence, combinatorial inhibition of mTOR and GSK3β mTOR inhibition, activation of GSK3β in response to may synergistically suppress tumorigenesis. PI3K/mTOR inhibition may lead to the resistance to PI3K/mTOR inhibitors in PIK3CA-mutated cancer [176]. mTOR mutations A recent study demonstrates that lung squamous cell Gene mutations may affect the sensitivity of a drug that carcinoma adapt to chronic mTOR inhibition through the targets the protein encoded by this gene. More than 30 GSK3α/β signaling pathway, which involves the metabolic activating mutations of mTOR have been reported in reprogramming via increased glutaminolysis [177]. One human cancer, such as L1460P, C1483F, E1799K, F1888L, study also reveals that glutaminase (GLS) and glutamate T1977R, V2006I, V2046A, S2215Y, L2230V, E2388Q, levels are elevated in glioblastoma after treating with I2500F, R2505P, and D2512H [127, 161]. Cancer cells that mTOR inhibitor [178]. Treatment with GSK3 inhibitors harbor a subset of those mutations, including C1483F, or the glutaminase inhibitor effectively overcomes the E1799K, and S2215Y, are hypersensitive to rapamycin, resistance to mTOR inhibition [176–178]. Moreover, the whereas three mutations (A2034V, F2018L, and S2035F) activation of the purine salvage pathway due to increased in the FRB domain of mTOR are associated with rapa- expression of hypoxanthine phosphoribosyl transferase 1 mycin resistance [162, 163]. While tumor cells with muta- leads to the resistance to the dual PI3K/mTOR inhibitor tions in the kinase domain are still responsive to rapalogs gedatolisib [179]. In fact, mTOR is tightly involved in [161], mutations in the kinase domain of mTOR, such as purine metabolism. mTORC1 is not only activated by M2327I, S2215Y, L2230V, E2388Q, and V2046A, may be purine nucleobases or nucleosides [49], but also promotes responsible for the resistance to the ATP-competitive purine synthesis by ATF4-mediated upregulation of the inhibitor MLN0128 [127]. It remains to know whether mitochondrial tetrahydrofolate (mTHF) cycle enzyme activating mutations in the kinase domain of mTOR are methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) responsible for the resistance to allosteric mTOR kinase [180]. Moreover, mTORC1 promotes de novo pyrimi- inhibitors other than MLN0128. In addition, there are dine biosynthesis by S6K1-mediated phosphorylation recurrent mutations in other mTOR pathway genes, such of carbamoyl-phosphate synthetase 2, aspartate trans- as raptor, rictor,and RHEB [163]. RHEB-Y35N mutant carbamylase, and dihydroorotase (CAD) [181, 182]. gains the function to activate mTORC1 [161]. It warrants Therefore, the increased expression of hypoxanthine further studies to clarify which cancer-associated muta- phosphoribosyl transferase 1 may rescue the defect in tions in raptor, rictor, and RHEB may be associated with purine synthesis due to mTOR inhibition and help cancer mTOR inhibitors resistance. cells adapt to mTOR inhibition. Another compensatory response to mTORC1 inhibition Rewiring of oncogenic or metabolic pathways is the upregulation of transglutaminase 2, a multifunc- The sensitivity to mTOR inhibitors is regulated by other tional enzyme that is involved in cross-linking polypeptide oncogenic pathways, such as PI3K, MAPK, AURKA, and chains with e-(c-glutamyl)-lysine, apoptosis, signal trans- NF-kB signaling [164, 165]. Both the Ras/MAPK and duction, cell migration, cell adhesion, and extracellular Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 10 of 19 matrix remodeling [183–185]. Inhibition of transglutami- (PFS) among patients with progressive advanced pancreatic nase 2 potently sensitizes mTORC1-hyperactive cancer neuroendocrine tumors [197]. As registered in clinical- cells to rapamycin in vitro and in vivo [183]. Moreover, trials.gov,there aremorethan80clinicaltrialsfor mTOR mitochondria homeostasis is critical for cell growth and inhibitor monotherapy in cancer patients. A phase 2 trial survival. Mitochondrial hyperfusion is an adaptive of everolimus in patients with recurrent adult low-grade response to mTOR inhibition. Mechanistically, the gliomas demonstrates a high degree of disease stability translation of mitochondrial fission process 1 (MTFP1) [198]. Moreover, everolimus has a promising effect in is suppressed by mTOR inhibitors, which eventually patients with heavily pretreated, relapsed, or refractory results in mitochondrial hyperfusion, a process that classical Hodgkin’s lymphoma, with an overall response antagonizes apoptosis [186]. rate (ORR) of 45.6%, a median PFS of 8 months, and a long-term response (≥ 12 months) rate of 12% [188]. Of Clinical testing of mTOR inhibitors note, everolimus exhibits clinical activity as the first-line Given that preclinical studies demonstrate the anti-cancer monotherapy in a phase 2 clinical trial in 27 patients with efficacy of mTOR inhibitors alone or in combination with advanced biliary tract cancer [199]. Another phase 2 clin- chemotherapy, radiotherapy, and targeted therapy, there ical trial in 35 patients with thyroid cancer demonstrates are many completed or ongoing clinical trials to test the that everolimus has clinical benefit in patients with efficacy of mTOR inhibitors for treating various types of advanced differentiated thyroid cancer [200]. Also, single- human cancer (Table 1). In general, most of mTOR in- agent ridaforolimus has anti-tumor activity and acceptable hibitors are well tolerated, while there are some common tolerability in advanced endometrial cancer patients [201]. adverse effects including fatigue, rash, mucositis, and These observations need to be validated in a large scale of metabolic complications. mTOR inhibitors are associated randomized clinical trials. with a significantly increased risk of hyperglycemia, Based on a phase 2 trial in 167 patients, oral administra- hypertriglyceridemia, and hypercholesterolemia [187]. tion of the mTOR kinase inhibitor voxtalisib (50 mg, twice Other adverse events of everolimus are thrombocytopenia, daily) exhibits a promising efficacy in patients with folli- anemia, nausea, and stomatitis [188]. Ridaforolimus is cular lymphoma but limited efficacy in patients with orally bioavailable and better tolerated in children than mantle cell lymphoma, diffuse large B cell lymphoma, or the adults [189]. Deforolimus was well tolerated and chronic lymphocytic leukemia/small lymphocytic lym- showed encouraging anti-tumor activity across a broad phoma [202]. Of note, serious adverse events occurred in range of malignancies when administered intravenously, 58.1% of patients [202]. In contrast, the clinical efficacy of and a dose of 12.5 mg/day is being evaluated in phase II MLN0128 in patients with metastatic castration-resistant trials [190]. prostate cancer is limited, possibly due to the dose re- Moreover, MLN0028-treated patients may suffer from ductions secondary to toxicity [191]. Although it is anorexia, dyspenea and macunopapular rash [191]. In expected that mTOR kinase inhibitor may have superior clinical trials of solid tumors, the PI3K/mTOR inhibitor efficacy than rapalogs, a randomized phase 2 trial in NVP-BEZ235 (twice daily) is poorly tolerated, which patients with metastatic clear cell renal cancer demon- leads to treatment discontinuation in some patients and strated that the PFS and OS of AZD2014 were less than limits its efficacy in treating cancer [192, 193]. Apitolisib that of everolimus [203]. While the PI3K/mTOR inhibitor (GDC-0980), another dual pan-PI3K/mTOR inhibitor, NVP-BEZ235 is poorly tolerated in cancer patients, a also has grade 3–4 adverse effects and is less effective clinical trial in patients with recurrent endometrial cancer than everolimus [194]. GSK2126458 (GSK458) plus demonstrated that weekly intravenous administration of trametinib has poor tolerability, due to skin and gastro- another P3K/mTOR inhibitor gedatolisib achieved mo- intestinal toxicities such as diarrhea [195]. Daily oral derate anti-cancer activity with tolerable toxicity [204]. administration of PF-04691502 (8 mg/day) has adverse events including fatigue, nausea, vomiting, hypergly- mTOR inhibitors in combination therapy cemia, and rash [196]. The occurrence of the above- While mTOR inhibitor monotherapy has efficacy in some mentioned adverse effects following treatment with type of cancer, preclinical studies demonstrate strong mTOR inhibitors may be due to the critical roles of rationales for combinatorial treatment with mTOR in- mTOR in metabolism and immunity. hibitors and other drugs. For example, inhibition of both Akt/mTOR and WNT/β-catenin pathways synergistically mTOR inhibitors monotherapy suppresses AML [205]. As registered in clinicaltrials.gov, Everolimus has been approved by the FDA for the treat- there are many clinical trials to test the efficacy of mTOR ment of advanced renal cell carcinoma, pancreatic inhibitors in combination with other molecular targeted neuroendocrine tumors, and advanced breast cancer [83]. or chemotherapeutic agents. For example, everolimus is Everolimus significantly improves progression-free survival combined with one or several chemotherapeutic agents, Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 11 of 19 Table 1 Clinical evaluation of mTOR inhibitors mTOR inhibitor Category Combination Cancer type Phase Response PFS (months) OS (months) Ref. or trial ID* Everolimus (RAD001) Rapalog None Thyroid cancer 2 No CR/PR; SD (> 24 weeks) 9 (95% CI 4–14) 18 (95% CI 7–29) 200 58% Everolimus Rapalog Letrozole Relapsed ER(+) high- 2 CR 0; PR 16%; SD 37% 3.9 (95% CI 2.8–11); 13; 6-month OS rate, 209 grade ovarian cancer 3-month rate, 47%; 84% 6-month rate, 32% Everolimus Rapalog Exemestrane ER(+) locally advanced 3 CBR 33.4% vs 18% (control; 6.93 (95% CI 6.44–8.05) 30.98 (95% CI 27.96–34.56) NCT00863655 or metastatic breast placebo plus exemestrane) vs 2.83 (95% CI 2.74–4.14) vs control 26.05 (95% cancer (placebo plus exemestrane) CI 22.57–33.08) Everolimus Rapalog None Advanced neuroendocine 3 Not available 11.04 (95% CI 8.41–13.86) vs 44.02 (95% CI 35.61–51.75) vs NCT00510068 tumor placebo 4.6 (95% placebo 37.68 (95% CI 3.06–5.49) CI 29.14–45.77) Everolimus Rapalog Rituximab Diffuse large B cell 2 ORR 38% (90% CI 21–56%); 2.9 (90% CI 1.8–3.8) 8.6 (90% CI 4.9–16.3) 212 lymphoma CR 3/24; PR 6/24 NCT00869999 MLN0128 ATP-competitive Paclitaxel and Advanced solid tumors 1 CR 0; PR 8/54; SD (> 6 months) Not available Not available 87 trastuzumab 6/54 NCT01351350 AZD2014 (Vistusertib) ATP-competitive None Metastatic clear cell renal 2 Response rate 4% for AZD1024, 1.8 vs 4.6 for everolimus 4.9 for AZD1024 203 cancer 13% for everolimus treatment Progressive disease 69% vs 13% for everolimus treatment Voxtalisib (SAR24540; ATP-competitive None Relapsed or refractory 2 CR 8/164 (4.9%); PR 22/164 1.9 for follicular lymphoma Not available 202 XL765) non-Hodgkin lymphoma or (13.4%); Overall progression-free NCT01403636 chronic lymphocytic SD 55/164 (33.5%); ORR 18.3% rate at 24 weeks, 38·6% lymphoma (40.3% for follicular lymphoma) (95% CI 30·9–46·3) Gedatolisib (PKI-587; ATP-competitive None Recurrent endometrial 2 CR 1/38 (3%); PR 5/38 (13%); 3.7 (95% CI 2–5.6) for Not available 204 PF05212384) cancer SD > 16 weeks, 24% (37% for stathmin-low cancer; 3 NCT01420081 stathmin-low cancer, 11% for (95% CI 1.87–5.7) for stathmin-high cancer) stathmin-high cancer CR complete response, CBR clinical benefit rate, ORR overall response rate, OS overall survival, PFS progression-free survival, PR partial response, SD stable disease. *, Registration number in ClinicalTrials.gov Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 12 of 19 such as taxol, cisplatin, carboplatin, oxaliplatin, irinotecan, humanized monoclonal antibody against IGF-1R, and temozolomide, and gemcitabine. temsirolimus was tested in a clinical setting. This com- The phase 3 BOLERO-2 trial in patients with ER- bination shows clinical activity in patients with sarcoma positive/HER2-negative advanced or metastatic breast and adrenocortical carcinoma [215, 216]. In addition, a cancer demonstrates that a combination of everolimus combination of everolimus (5 mg daily) and the multi- and the aromatase inhibitor exemestane significantly im- kinase inhibitor sorafenib (400 mg twice daily) exhibits proves PFS, while the OS is not improved [206, 207]. Ac- anti-tumor activity in previously untreated patients with cordingly, a combination of everolimus and exemestane metastatic renal cell carcinoma with tolerable toxicity has been approved as a guideline for treating ER-positive/ [217]. However, a combination of sorafenib and evero- HER2-negative advanced or metastatic breast cancer limus fails to achieve the target of 6 month PFS of 50% [208]. In a phase 2 clinical trial, a combination of everoli- or greater among patients with unresectable high-grade mus and the aromatase inhibitor letrozole achieved a 12- osteosarcoma progressing after standard treatment week PFS rate of 47% in patients with ER-positive relapsed [218]. For patients with recurrent glioblastoma, a com- high-grade ovarian cancer [209]. In addition, the combin- bination of sorafenib (200 mg twice daily) and temsiroli- ation of everolimus with trastuzumab and paclitaxel has a mus (20 mg weekly) is associated with considerable promising efficacy in patients with highly resistant HER2- toxicity and poor efficacy [219]. positive advanced breast cancer (Table 1). This combin- In patients with metastatic castration-resistant prostate ation is currently under investigation in the BOLERO-1 cancer, a combination of everolimus and the EGFR phase 3 trial [210]. Moreover, a combination of everolimus inhibitor gefitinib has no significant anti-tumor activity with carboplatin is efficacious in treating metastatic triple- [220]. According to a phase 2 trial, a combination of negative breast cancer, with a median PFS of 3 months sunitinib and everolimus as the first-line therapy exhibits (95% CI 1.6 to 4.6 months) and overall survival (OS) of poor efficacy in treating advanced renal cell carcinoma 16.6 months [211]. In contrast, a combination of everoli- [221]. However, another phase 2 trial in patients with mus with gemcitabine/cisplatin has no synergistic effect in metastatic renal carcinoma demonstrates that the first- patients with metastatic triple-negative breast cancer. line sunitinib treatment followed by everolimus achieves Hence, this combination still needs validation in more a longer OS than the first-line everolimus followed by patients. sunitinib, suggesting that the sequence may affect the The CD20-targeted monoclonal antibody rituximab is a outcome [222]. Moreover, a combination of imatinib and treatment for low-grade or follicular CD20-positive non- everolimus has limited activity in the treatment of pa- Hodgkin’s lymphoma. Diffuse large B cell lymphoma tients with advanced chordoma [223]. The combination (DLBCL) is the most common type of non-Hodgkin’s of pimasertib and voxtalisib showed a poor long-term lymphoma. A phase 2 study of everolimus (10 mg/day) in tolerability and limited anti-tumor activity in patients combination with rituximab demonstrated an overall with advanced solid tumors [224]. response rate of 38%, a complete response rate of 12.5%, and a partial response rate of 25% among 24 patients with heavily pretreated DLBCL [212]. In addition, the combi- Concluding remarks nation of everolimus with rituximab or rituximab plus The discovery of TOR in yeast and mTOR in mammals cyclophosphamide, doxorubicin, vincristine, and pred- is a fundamental breakthrough in understanding cell and nisone (R-CHOP) was well tolerated in DLBCL patients organism growth, metabolism, and diseases. In-depth [212, 213]. It warrants further study to determine if the studies to clarify the regulators and effectors of mTOR combination of everolimus with R-CHOP has a better signaling have revealed multiple networks that work to- response in patients with DLBCL. In addition, the gether to integrate growth factors, nutrients, sterols, and combination of mTORC1/2 inhibitor with other targeted nucleotides signaling. The identification of the critical cancer drugs has been tested in clinical trials. Among 54 roles of mTOR and its regulators in tumorigenesis has cancer patients who were treated with MLN0128 and driven the development of the ever-growing list of trastuzumab/paclitaxel, 14.8% (8/54) of them achieved a mTOR inhibitors. While some of the mTOR inhibitors partial response, and near 11% (6/54) cases had stable have been approved to treat cancer patients, more disease for more than 6 months [87]. According to a phase mTOR inhibitors are under check to fulfill their promise 1 trial (NCT02193633), the combination of paclitaxel and for cancer therapy. vistusertib is highly active and well tolerated in patients It appears that mTOR inhibitors have mixed efficacy with high-grade serous ovarian cancer and squamous in patients with distinct kinds of cancer and among non-small cell lung cancer [214]. patients with the same kind of cancer. Recent studies Given that IGF-IR signaling may induce mTORC1 reveal that tumor organoids may help drug testing inhibitor resistance, the combination of cixutumumab, a [225, 226]. Tumor organoids may be used to test the Hua et al. Journal of Hematology & Oncology (2019) 12:71 Page 13 of 19 response of a given tumor to mTOR inhibitors. Alter- lethal with SEC13 protein 8; mSIN1: Mammalian stress-activated protein kinase-interacting protein 1; mTOR: Mechanistic target of rapamycin; natively, patient-derived tumor grafts may be transplanted PI3K: Phosphoinositide 3-kinase; PKC: Protein kinase C; PRAS40: 40 kDa to animals, followed by testing their response to mTOR proline-rich Akt substrate; Raptor: Regulatory-associated protein of mTOR; inhibitors [227]. It would be of interest to determine if these RCC: Renal cell carcinoma; RHEB: Ras homolog enriched in the brain; Rictor: Rapamycin-insensitive companion of mTOR; S6K1: Ribosomal protein emerging technologies are clinically relevant. S6 kinase β-1; TSC: Tuberous sclerosis complex In the era of precise medicine, it needs to determine if there are predictive biomarkers that may guide the Acknowledgements We would like to thank Qiulin Tang for her assistance in preparing the stratification of patients in clinical trials or help identify manuscript. the patients who most likely benefit from treatment with mTOR inhibitors in a clinical setting. Gene testing is a Authors’ contributions promising approach to achieve this goal. The candidates HH and YJ conceived the review and wrote the manuscript. QK and JW prepared the figures. HZ edited the references. TL was involved in editing for gene testing may include mTOR, PIK3CA, GATOR, the manuscript. All authors read and approved the final manuscript. KRAS, and BRAF. Mutations in PIK3CA and GATOR have been associated with higher sensitivity to mTOR Funding inhibition in preclinical studies. Hence, PIK3CA muta- This work was supported by grants 81672814 and 81872388 from the National Natural Science Foundation of China and grant 2018SCUH0009 tions may be potential sensitive markers. In contrast, from the Fundamental Research Fund for the Central Universities. KRAS/BRAF mutations may be resistant biomarkers. Both DNA from tumor samples and ctDNA from the Availability of data and materials Not applicable. blood may be subject to testing of gene mutations. In addition, gene mutations in the tumors may be dynamic Ethics approval and consent to participate during cancer evolution or regression [228]. It remains Not applicable. to determine if dynamic testing of ctDNA during the Consent for publication course of therapy may monitor cancer evolution and Not applicable. better predict drug resistance, thereby adjusting the treatment regimen in time. Recent progress in liquid Competing interests biopsy may help address this critical issue [229, 230]. In The authors declare that they have no competing interests. addition to gene testing, the solvable factors in the Author details blood may be potential biomarkers as well. Of particu- State Key Laboratory of Biotherapy, Laboratory of Stem Cell Biology, lar note, the mechanisms underlying the varied res- National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu 610041, China. Laboratory of Oncogene, Cancer Center, ponsiveness to mTOR inhibitors in cancer patients may West China Hospital, Sichuan University, Chengdu, China. School of Basic be complex. Rather than a single or few biomarkers, a Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, set of biomarkers may be more powerful and accurate China. Cancer Center, West China Hospital, Sichuan University, Chengdu, China. to meet the challenge. 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